Objective To discuss the effect of different local pulse vibration stimulation treatment conditions on the recovery of proprioception after anterior cruciate ligament reconstruction （ACLR） in the rabbits，and to provide the reference for clinical practice. Methods A total of 45 healthy New Zealand white rabbits were selected，15 rabbits were used to prepare the allogenic tendon grafts， and 30 rabbits were used to prepare the anterior cruciate ligament （ACL） model. After modeling， the rabbits were randomly divided into control group and nine vibration stimulation treatment groups （vibraory stimulation treatment groups 1—9）， and there were three rabbits in each group. Orthogonal experiment was used to design the vibration stimulation treatment parameters， and the ACL samples of the rabbit in various groups were acquired； HE staining was used to observe the morphology of the proprioceptors of the rabbits in various groups； S100 immunohistochemistry staining was used to observe the types of proprioceptors of the rabbits in various groups； the number of proprioceptive cells of the rabbits in various groups was counted； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression level of growth associated protein-43 （GAP-43） mRNA in ACL tissue of the rabbits in various groups； orthogonal analysis method was used to select the optimal parameters for the recovery vibration stimulation treatment after ACLR. Results The graft tension was satisfactory， without slack state，with negative front drawer and Lachman tests；the continuity of graft could be seen in ACL tissue of the rabbits， healing with the tibial and femoral tendons. The Ruffini corpuscles in the ACL proprioceptor were oval or dendritic， with the diameters of 25—330 μm； the pacinian corpuscles showed round or elliptical sensors， with the diameters of 40—220 μm； the Golgi tendon organs showed fusiform， with the diameters of 140—900 μm； the free nerve endings were myelin-free nerve endings， with the diameters of 0.5—1.5 μm. The proprioceptors in the transplanted ACL were mainly distributed in the tibial and femoral attachment points of the ACL， mainly Ruffini-like corpuscles and Pacinian-like corpuscles，without typical Golgi-like tendon organs. Compared with control group， the numbers of proprioceptive cells in vibratory stimulation treatment groups 3， vibratory stimulation treatment 5， and vibratory stimulation treatment 7 groups were significantly increased （F=28.49， P<0.01）. There was no significant difference in the expression of GAP-43 mRNA in the cells between various groups （F=0.83， P>0.05）. The amplitude intensity， vibration frequency，and action time had a significant effect on the number of proprioceptors in the treatment of local pulse vibration stimulation after ACLR，and the effect degree was amplitude intensity>action time>vibration frequency. The optimal amplitude intensity was 3 mm， the optimal vibration frequency was 25 Hz， and the best action time was 10 min. Conclusion The local pulse vibration stimulation treatment can promote the recovery of ACL proprioceptors in the rabbits after ACLR. Low amplitude， low vibration frequency， and short duration of local pulse vibratory treatment could be used to accelerate the recovery of proprioception after ACLR.

Objective To discuss the protective effect of fructus mume total flavonoids （FMF） on the injury of the SH-SY5Y cells induced by 1-methyl-4-phenylpyridinium（MPP⁺） through regulating microRNA （miR）-145-3p expression，and to clarify the mechanism. Methods The SH-SY5Y cell model of Parkinson’s disease （PD） was established by MPP⁺ induction. The optimal intervention concentration and action time of MPP⁺ and FMF were screened out by CCK-8 assay. The cells were divided into control group （untreated）， model group （treated with 500 μmol·L^－1 MPP⁺ for 24 h）， MPP⁺+mimics group （transfected with miR-145-3p mimics）， MPP⁺+NC group （transfected with miR-145-3p NC）， MPP⁺+FMF group treated with 0.25 g·L^－1 FMF for 24 h）， MPP⁺+mimics+FMF group （transfected with miR-145-3p mimics and treated with 0.25 g·L^－1 FMF for 24 h），and MPP⁺+NC+FMF group （transfected with miR-145-3p NC，and （treated with 0.25 g·L^－1 FMF for 24 h）. CCK-8 assay was used to detect the proliferation abilities of the cells in various groups； Annexin Ⅴ-FITC/PI staining was used to the detect the apoptotic rates of the cells in various groups； transmission electron microscope was used to observe the ultrastructure morphology of mitochondrial autophagy in the cells in various groups； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of miR-145-3p in the cells in various groups； Western blotting method was used to detect the expression level of Beclin-1 protein and the ratio of LC3-Ⅱ/ LC3-Ⅰ in the cells in various groups. Results After treated with different concentrations of MPP⁺， the proliferation ability of the cells was significantly decreased（P<0.01）， and the PD cell model was successfully constructed. The optimal concentrations and action time of MPP⁺ and FMF intervention were 500 μmol·L^－1， 24 h， and 0.25 g·L^－1，24 h， respectively.The CCK-8 assay results showed that the proliferation ability of the cells in model group was significantly lower than that in control group （P<0.01）； the proliferation abilities of the cells in MPP⁺+mimics group and MPP⁺+FMF group were significantly higher than that in model group（P<0.01）； the proliferation ability of the cells in MPP⁺+mimics+FMF group was significantly higher than that in MPP⁺+FMF group （P<0.01）. The Annexin V-FITC/PI staining results showed that the apoptotic rate of the cells in model group was significantly higher than that in control group （P<0.01）； the apoptotic rates of the cells in MPP⁺+mimics group and MPP⁺+FMF group were significantly lower than that in model group （P<0.01）； the apoptotic rate of the cells in MPP⁺+mimics+FMF group was significantly lower than that in MPP⁺+FMF group （P<0.01）. The transmission electron microscope results showed that the cells in control group showed the uniform and intact mitochondrial morphology， the cells in model group showed the enlarged and irregular mitochondria with vacuolar changes，and the cells in MPP⁺+mimics group and MPP⁺+FMF group showed the improved mitochondrial structure， compared with model group， the autophagosome numbers of the cells in MPP⁺+mimics group and MPP⁺+FMF group were increased； the cells in MPP⁺+mimics+FMF group showed more intact mitochondrial structure， compared with MPP⁺+FMF group， the autophagosome numbers in the cells was increased. The RT-qPCR results showed that the expression level of miR-145-3p in the cells in model group was significantly lower than that in control group （P<0.01）； the expression levels of miR-145-3p in the cells in MPP⁺+mimics group and MPP⁺+FMF group were significantly higher than that in model group （P<0.01）； the expression level of miR-145-3p in the cells in MPP⁺+mimics+FMF group was significantly higher than that in MPP⁺+FMF group （P<0.01）.The Western blotting results showed that the expression level of Beclin-1 protein and the ratio of LC3-Ⅱ/LC3-Ⅰ in the cells in model group were lower than those in control group （P<0.05）； the expression level of Beclin-1 protein and the ratio of LC3-Ⅱ/ LC3-Ⅰ in the cells in MPP⁺+mimics group and MPP⁺+FMF group were significantly higher than those in model group （P<0.01）； the expression level of Beclin-1 protein and the ratio of LC3-Ⅱ/ LC3-Ⅰ in the cells in MPP⁺+mimics+FMF group were significantly higher than those in MPP⁺+FMF group （P<0.01）. Conclusion FMF may promote the autophagy， alleviate the mitochondrial dysfunction， and attenuate the injury of the SH-SY5Y cells induced by MPP⁺， and its mechamism is related to upregulating the miR-145-3p expression.

Objective To construct the sex-determined region Y-box 17 （SOX17） over-expression lentiviral vector， and to construct the cell line stably over-expressing SOX17 by using the PC12 cells infected with SOX17 over-expression lentivirus. Methods The over-expression sequence of SOX17 was designed and synthesized based on the National Center for Biotechnology Information （NCBI） Database， and was connected to the GV492 lentiviral vector after being doubly digested with BamHⅠ and AgeⅠ enzymes to construct the GV492-SOX17 over-expression recombinant plasmid. The agarose gel electrophoresis was used to identify the PCR products， and the positive bacteria carrying the GV492-SOX17 over-expression recombinant plasmid were selected， cloned and sequenced. The GV492 empty plasmid and GV492-SOX17 over-expression recombinant plasmid were transfected into the human embryonic kidney HEK 293T cells， respectively. After transfected for 48 h， the GV492 control lentivirus and GV492-SOX17 over-expression lentivirus were packaged and the viral titer was detected. The PC12 cells were divided into blank group， GV492 control group， and GV492-SOX17 group. The cells in blank group was not treated， and the cells in GV492 control and GV492-SOX17 groups were infected with the corresponding lentivirus （multiplicity of infection = 100）， and the PC12 cells successfully infected with lentivirus were selected with 10 mg·L^－1 puromycin. The growth state and green fluorescence expression of the PC12 cells were observed under fluorescence microscope；real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression level of SOX17 mRNA in the PC12 cells in various groups；Western blotting method was used to detect the expression level of SOX17 protein in the PC12 cells in various groups. Results The gene fragment length of GV492-SOX17 over-expression recombinant plasmid was about 744 bp， and the sequence of GV492-SOX17 over-expression recombinant plasmid gene was identical to the designed and synthesized SOX17 over-expression sequence. The titers of GV492 control lentivirus and GV492-SOX17 over-expression lentivirus were both 2.5×108 TU·mL^－1. The growth state of the PC12 cells in various groups was good and the green fluorescence was expressed. The RT-qPCR results showed that the expression level of SOX17 mRNA in the cells in GV492-SOX17 group was significantly higher than those in blank group and GV492 control group（P<0.01）. The Western blotting results showed that there was a specific band appeared at a relative molecular mass of 44 000， suggesting the SOX17 protein was successfully expressed in the PC12 cells； compared with blank group and GV492 control group， the expression level of SOX17 protein in the cells in GV492-SOX17 group was increased （P<0.05）. Conclusion The GV492-SOX17 lentiviral expression vector is successfully constructed and the PC12 cell line stably over-expressing GV492-SOX17 is established.

Objective To discuss the effect of head acupuncture on the neurological function and the expressions of hypoxia-inducible factor （HIF）-1α and vascular endothelial growth factor receptor 2 （VEGFR2） in the brain tissue of rats with focal cerebral ischemia， and to clarify the related mechanism. Methods The modified middle cerebral artery occlusion（MCAO） method was used to prepare the focal cerebral ischemia rat models.Twenty successfully modeling rats were randomly divided into model group and treatment group， and there were 10 rats in each group；another 10 SD rats were selected as sham operation group.The rats in treatment group were treated with scalp acupuncture. The neurological function scores of the rats in various groups were recorded and the model was verified； Morris water maze test was used to detect the learning and memory functions of the rats in various groups； enzyme-linked immunosorbent assay （ELISA） method was used to detect the level of HIF-1α in serum and brain tissue of the rats in various groups；Western blotting method was used to detect the expression levels of HIF-1α and VEGFR2 proteins in brain tissue of the rats in various groups. Results The neurological function scores detection results showed that there was no neurological deficit of the rats in sham operation group； compared with sham operation group， the neurological function scores of the rats in model group and treatment group were increased （P<0.05）. The Morris water maze test results showed that compared with sham operation group， the time to find the platform of the rats in model group was increased （P<0.05）； compared with model group， the time to find the platform of the rats in treatment group was decreased （P<0.05）. The ELISA results showed that compared with sham operation group， the levels of HIF-1α in serum and brain tissue of the rats in model group were increased （P<0.05）；compared with model group， the levels of HIF-1α in serum and brain tissue of the rats in treatment group were increased （P<0.05）. The Western blotting results showed that compared with sham operation group， the expression levels of HIF-1α and VEGFR2 proteins in brain tissue of the rats in model group were increased （P<0.05）； compared with model group， the expression levels of HIF-1α and VEGFR2 proteins in brain tissue of the rats in treatment group were increased （P<0.05）. Conclusion The head acupuncture may restore the function of brain tissue in the rats with focal cerebral ischemia and up-regulate the levels of HIF-1α and VEGFR2 in brain tissue of the rats， and its related mechanism may be related to activating the HIF/VEGFR2 angiogenesis pathway.

Objective To discuss the effect of ligustrazine on growth of the glioma stem cells（GSCs）subcutaneous xenografts in the nude mice， transforming growth factor-β （TGF-β） signaling pathway and epithelial-mesenchymal transition（EMT），and to provide the reference for selecting drugs in the clinical therapy of glioma. Methods The human brain glioma U87 cells were collected and the GSCs were cultured， separated， enriched， and identified. Forty female BALB/c nude mice were used to establish the GSCs subcutaneous xenograft models， which was randomly divided into model group and low， medium， and high doses of ligustrazine groups， and there were 10 mice in each group. The mice in low， medium， and high doses of ligustrazine groups were intraperitoneally injected with ligustrazine once every 3 d， and the dosages were 1.5， 3.0， and 6.0 mg·kg^－1， respectively， while the mice in model group were only given equal volume of saline，and this process continued for 15 d. The transplanted tumor volumes and tumor weights were detected， and the inhibitory rates of tumor weights of the mice was caculated. The tumor growth curve was drawn.HE staining was used to observe the pathomorphology of tumor tissue of the mice；real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of TGF-β1 and transforming growth factor β receptor Ⅰ（TβRⅠ） mRNA in tumor tissue of the mice in various groups； Western blotting method was used to detect the expression levels of TGF-β1， TβRⅠ，small mother against decapentaplegic homolog 2/3 （Smad2/3）， phosphorylated Smad2/3 （p-Smad2/3），E-cadherin， TWIST family bHLH transcription factor 1 （TWIST1），Vimentin， and Snail proteins in tumor tissue of the mice in various groups. Results The GSCs were successfully enriched and separated from the U87 cells， and the immunofluorescence staining resutls showed the positive expression of stem cell marker CD133. Compared with model group， the transplanted tumor volumes of the nude mice in low， medium， and high doses of ligustrazine groups were significantly decreased （P<0.05）， in a dose-dependent manner； the tumor weights were also significantly decreased （P<0.05） in a dose-dependent manner as well. The inhibitory rates of tumor weights in low， medium， and high doses of ligustrazine groups were 12.72%， 39.90%， and 55.36%， respectively.The HE staining results showed that the cells in tumor tissue of the mice in model group were gradually increased and exhibited good morphology and tight connection， the growth density was high， and the atypical nuclei was obviously； the tumor tissue structures of the mice in low， medium， and high doses of ligustrazine groups were disordered， the cell density was gradually decreased， the nuclear condensation and karyorrhexis were increased，the necrotic zones were gradually enlarged， and these improvements became more obvious as the increasing of the dose of ligustrazine. The RT-qPCR results showed that compared with model group， the expression levels of TGF-β1 and TβRⅠ mRNA in tumor tissue of the mice in low， medium， and high doses of ligustrazine groups were decreased （P<0.05） in a dose-dependent manner.The Western blotting results showed that compared with model group， the expression levels of E-cadherin protein in tumor tissue of the mice in low， medium， and high doses of ligustrazine groups were increased （P<0.05），and the expression levels of TWIST1，Vimentin，Snail，TGF-β1，TβRⅠ，and p-Smad2/3 proteins were decreased（P<0.05） in a dose-dependent manner. Conclusion Ligustrazine can inhibit the growth of GSCs subcutaneous xenografts in the nude mice， and can interfere the TGF-β1/Smad2/3 signaling activation and EMT induction.

Objective To discuss the effect of usnic acid on the proliferation， apoptosis， migration， and invasion of the hypertrophic scar fibroblasts （HSFBs）， and to clarify its regulatory effect on the c-Jun N-terminal kinase （JNK）/mitogen-activated protein kinase （MAPK） signaling pathway. Methods The HSFBs at logarithmic growth phase were divided into control group， 10 μmol·L^－1 usnic acid group， 20 μmol·L^－1 usnic acid group， and 50 μmol·L^－1 usnic acid group. Except for control group， the cells in the other groups were treated with the corresponding concentrations of usnic acid. The survival rates of the cells in various groups were detected by methylthiazolyidiphenyl-tetrazolium bromide（MTT） method； the apoptotic rates of the cells in various groups were detected by flow cytometry；the invasion and migration abilities of the cells in various groups were detected by Transwell chamber experiment；the expression levels of phosphorylated JNK （p-JNK）， JNK， B-cell lymphoma-2（Bcl-2）， and Bcl-2-associated X protein （Bax） proteins in the cells in various groups were detected by Western blotting method. Results The MTT and flow cytometry results showed that compared with control group， the survival rates of the cells in 10， 20， and 50 μmol·L^－1 usnic acid groups were decreased （P<0.05）， and the apoptotic rates were increased （P<0.05）. Compared with 50 μmol·L^－1 usnic acid group， the survival rates of the cells in 10 and 20 μmol·L^－1 usnic acid groups were decreased （P<0.05）， and the apoptotic rates were increased （P<0.05）.The Transwell chamber experiment results showed that compared with control group，the numbers of invasion and migration cells in 10，20 and 50 μmol·L^－1 usnic acid groups were decreased（P<0.05）； compared with 10 μmol·L^－1 usnic acid group，the numbers of invasion and migration cells in 20 and 50 μmol·L^－1 usnic acid groups were decreased（P<0.05）；compared with 20 μmol·L^－1 usnic acid group，the numbers of invasion and migration cells in 50 μmol·L^－1 usnic acid groups were decreased（P<0.05）.The Western blotting results showed that compared with control group，the expression levels of Bcl-2 protein in the cells in 10， 20， and 50 μmol·L^－1 usnic acid groups were decreased（P<0.05）， and the expression levels of Bax and p-JNK proteins were increased（P<0.05）；compared with 10 μmol·L^－1 usnic acid group，the expression levels of Bcl-2 protein in the cells in 20 and 50 μmol·L^－1 usnic acid groups were decreased， and the expression levels of Bax and p-JNK proteins in the cells were increased（P<0.05）； compared with 20 μmol·L^－1 usnic acid group，the expression level of Bcl-2 protein in the cells in 50 μmol·L^－1 usnic acid group was decreased（P<0.05）， and the expression levels of Bax and p-JNK proteins were increased（P<0.05）. Conclusion Usnic acid can inhibit the proliferation， invasion， and migration of the HSFBs， induce the apoptosis of the HSFBs， and activate the JNK/MAPK signaling pathway.

Objective To discuss the effect of polysaccharides from porphyra on the inflammatory factors secreted by macrophage RAW264.7 cells in the mice induced by lipopolysaccharide （LPS），and to clarify their immunomodulatory effect. Methods The RAW264.7 cells at logarithmic growth phase were divided into control group， LPS group， and LPS+polysaccharide from porphyra group. The viabilities of the cells in various groups were detected by CCK-8 method；the levels of interleukin-6 （IL-6），tumor necrosis factor-α （TNF-α）， monocyte chemotactic protein-1 （MCP-1）， macrophage inflammatory protein　（MIP）-1α，and MIP-1β in the cell culture supernatants in various groups were detected by enzyme-linked immunosorbent assay （ELISA） method. Results Compared with control group， the survival rate of the cells and the levels of IL-6， TNF-α， MCP-1， MIP-1α， and MIP-1β in the culture supernatant in LPS group and LPS+polysaccharide from porphyra group were significantly increased （P<0.01）. Compared with LPS group， the survival rate of the cells and the levels of IL-6， TNF-α， MCP-1， MIP-1α， and MIP-1β in the culture supernatant in LPS+polysaccharide from porphyra group were significantly decreased （P<0.01）. Conclusion polysaccharides from porphyra can inhibit the impact of the mouse macrophages on the secretion of inflammatory factors IL-6，TNF-α，MCP-1，MIP-1α，and MIP-1β，and have the potential immunomodulatory effect.

Objective To discuss the resistance and regeneration effects of long non-coding RNA （lncRNA） GPRC5D-AS1 on the muscle atrophy of myocytes in the mice induced by dexamethasone， and to clarify its mechanism. Methods The C2C12 cells at logarithmic growth phase were selected. The survival rates of the cells after treated with 0， 25， 50， 75， 100， 200 and 400 mg·L^－1 dexamethasone for 24， 48 and 72 h were detected， which was used to select the optimal dose and time to induce the muscle atrophy model.The expression level of lncRNA GPRC5D-AS1 in the C2C12 cells was detected by real-time fluroscence quantitative PCR（RT-qPCR） method to validate the cell model. The C2C12 cells were divided into normal group， control group，lncRNA GPRC5D-AS1-NC group，and lncRNA GPRC5D-AS1-OE group. The expression levels of lncRNA GPRC5D-AS1 in the cells in various groups were detected by RT-qPCR method. The levels of reactive oxygen species （ROS）， glutathione peroxidase 4 （GPX4）， ferrous ion （Fe²⁺） and malondialdehyde （MDA） in the cells in various groups were detected by flow cytometry and enzyme-linked immunosorbent assay （ELISA） method； the ultrastructural changes of mitochondria in the cells in various groups were observed under transmission electron microscope； the expression of myogenic differentiation protein （MyoD） in the cells in various groups was detected by immunofluorescence； the expression levels of ferroptosis-related proteins in the cells in various groups were detected by Western blotting method. Results The best modeling effect was achieved with 100 mg·L^－1 dexamethasone at 48 h. Compared with normal C2C12 cells， the expression level of lncRNA GPRC5D-AS1 in the C2C12 cells after treated with 100 mg·L^－1 dexamethasone for 48 h was decreased （P<0.05）， confirming that lncRNA GPRC5D-AS1 had a regulatory role in the muscle atrophy model. The RT-qPCR results showed that compared with normal group， the expression level of lncRNA GRPC5D-AS1 in the cells in model group was decreased （P<0.05）； compared with model group and lncRNA GRPC5D-AS1-NC group， the expression level of lncRNA GRPC5D-AS1 in the cells in lncRNA GRPC5D-AS1-OE group was increased （P<0.05）. The flow cytometry results showd that compared with normal group， the ROS level in the cells in model group was increased （P<0.05）； compared with model group and lncRNA GRPC5D-AS1-NC group， the ROS level in the cells in lncRNA GRPC5D-AS1-OE group was decreased （P<0.05）. The ELISA results showed that compared with normal group， the levels of Fe²⁺ and MDA in the cells in model group were increased （P<0.05）， and the level of GPX4 was decreased （P<0.05）； compared with model group and lncRNA GRPC5D-AS1-NC group， the levels of Fe²⁺ and MDA in the cells in lncRNA GRPC5D-AS1-OE group were decreased （P<0.05）， and the level of GPX4 was increased （P<0.05）. The transmission electron microscope results showed that the cells in normal group were round and normal in size with rich cilia on the membrane， and the mitochondria had a normal morphology； the cells in model group had less mitochondria and granulation in the cytoplasm， with blurred and swollen mitochondrial structures and typical ferroptosis-related manifestations； the number of mitochondria in the cells in lncRNA GRPC5D-AS1-OE group was increased，and the granulation in cytoplasm of the cells was decreased， and showed clearer mitochondrial structures. The immunofluorescence detection results showed that compared with normal group，the expression of MyoD in the cells in model group was decreased and the cells exhibited muscle atrophy； compared with model group and lncRNA GRPC5D-AS1-NC group， the expression of MyoD in the cells in lncRNA GRPC5D-AS1-OE group was increased， and the cells exhibited less muscle atrophy and more regeneration of myocytes. The Western blotting results revealed that compared with the normal group， the expression level of long-chain acyl-coenzyme A synthase （ACSL）4 protein in the cell in model group was increased （P<0.05）， and the expression levels of solute carrier family 7 member 11 （SLC7A11） and GPX4 proteins were decreased （P<0.05）； compared with model group and lncRNA GRPC5D-AS1-NC group， the expression level of ACSL4 protein in the cells in lncRNA GRPC5D-AS1-OE group was decreased （P<0.05）， and the expression levels of SLC7A11 and GPX4 proteins were increased （P<0.05）. Conclusion LncRNA GPRC5D-AS1 can protect the ferroptosis in the myocytes induced by dexamethasone and promote the repairment and regeneration of the cells，and its mechanism is related to regulating the SLC7A11/GPX4/ACSL4 signaling pathway.

Objective To discuss the effect of ganoderic acid A （GAA） on the proliferation， apoptosis， and migration ability of the PC-9 cells， and to clarify its mechanism. Methods The non-small cell lung cancer （NSCLC） PC-9 cells were cultured in vitro and divided into control group （without GAA）， low dose of GAA group （0.1 mmol·L^－1 GAA）， and high dose of GAA group （0.5 mmol·L^－1 GAA）. MTT method was used to detect the proliferation rates of the PC-9 cells in various groups； flow cytometry was used to detect the apoptotic rates of the PC-9 cells in various groups； scratch wound healing assay was used to detect the scratch healing rates of the PC-9 cells in various groups； Transwell chamber assay was used to detect the migration abilities of PC-9 cells in various groups； the expression levels of vascular endothelial growth factor （VEGF） and hypoxia-inducible factor-1α （HIF-1α） mRNA and protein in the PC9 cells in various groups were detected by real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting methods. Results The MTT assay results showed that compared with control group， the proliferation rate of the cells in low dose of GAA group was decreased after treated for 48 and 72 h （P<0.05）； after treated for 24， 48， and 72 h， the proliferation rate of the cells in high dose of GAA group was decreased （P<0.05）； compared with low dose of GAA group， the proliferation rate of the cells in high dose of GAA group was decreased after treated for 24， 48， and 72 h （P<0.05）.The flow cytometry results showed that compared with control group and low dose of GAA group， the apoptotic rate of the cells in high dose of GAA group was increased （P<0.05）. The cell scratch wound healing assay results showed that compared with control group and low dose of GAA group， the scratch wound healing rate of the cells in high dose of GAA group was decreased （P<0.05）. The Transwell chamber assay results showed that compared with control group and low dose of GAA group， the number of migration cells in high dose of GAA group was decreased （P<0.05）. The RT-qPCR and Western blotting results showed that compared with control group and low dose of GAA group， the expression levels of HIF-1α and VEGF mRNA and proteins in the cells in high dose of GAA group were decreased （P<0.05）. Conclusion High dose of GAA can inhibit the proliferation of the PC-9 cells and promote the apoptosis， and its mechanism may be related to regulating the expressions of VEGF and HIF-1α mRNA and proteins.

Objective To prepare the polypropylene carbonate（PPC）/polybutylene succinate（PBS） biofilm that mimics the structure of human bone periosteum，and to evaluate its physicochemical properties and biological characteristics. Methods The PPC/PBS biofilm was prepared by salting-out method. ¹H nuclear magnetic resonance spectra（¹HNMR） was used to observe the spectral absorption peak of PPC， PBS， and PPC/PBS，and the chemical structure changes of PPC， PBS， and PPC/PBS were analyzed； the ultramorphology of the PPC/PBS biofilm was observed by scanning electron microscope （SEM）； and the physicochemical properties， including porosity， Young’s modulus， fracture strength， fracture elongation， and static contact angle of the PPC/PBS biofilm were measured by GPC membrane permeation chromatography. The osteoblasts of the primary SD rat were isolated， cultured， and purified， and divided into control group （without biofilm）， BME-10X collagen membrane group， and PPC/PBS biofilm group. SEM was used to observe the adhesion and growth of the osteoblasts in various groups； cell counting was performed to detect the number of osteoblasts；alkaline phosphatase （ALP） assay kit was used to the differentiation of the osteoblasts； rabbit muscle degradation experiment was used to evaluate the degradation performance of the PPC/PBS biofilm in vivo. Results The PPC/PBS biofilm had a dual-layer structure consisting of a smooth surface layer and a rough surface layer， with a total thickness of approximately 0.5 mm and an average pore size of about 120 μm；the porosity was approximately 77.4%， the Young’s modulus was approximately 38.1 MPa， the fracture strength was approximately 1.22 MPa， the fracture elongation was approximately 7.4%， and the contact angle on the rough surface was 85°， while the contact angle on the smooth surface was 57°. The SEM observation results showed that fewer cells adhered to the surface of the PPC/PBS biofilm 1 d after culture； 3， 7， and 14 d after culture， a large number of osteoblasts were observed to adhere and grow on the surface of the biofilm， with cell protrusions attached to the film and cell bodies distributed in the pores. Compared with control group， the numbers of osteoblasts attached to the surface of the materials in BME-10X collagen membrane group and PPC/PBS biofim group were decreased after cultured for 1， 3， 7， and 14 d （P<0.05）. Compared with BME-10X collagen membrane group， the number of the osteoblasts attached to the surface of the materials in PPC/PBS biofilm group was increased after cultured for 1， 3， 7， and 14 d （P<0.05）. Compared with control group， the ALP levels in the osteoblasts attached to the surface of the materials in BME-10X collagen membrane group and PPC/PBS biofilm group were significantly decreased after cultured for 1， 3， 7， and 14 d （P<0.01）. Compared with BME-10X collagen membrane group， the ALP level in the osteoblasts attached to the surface of the materials in PPC/PBS biofilm group was significantly increased after cultured for 1 and 3 d （P<0.01）. The degradation experiment results showed that compared with 0 week after degradation， the weight loss rate and weight loss rate of number-avarage molecular of the PPC/PBS biofilm were increased 4 weeks after degradation （P<0.05）， while the fracture strength and fracture elongation were significantly decreased（P<0.05 or P<0.01）. From the 2nd week after degradation，the number of microstructures on rough surface of the PPC/PBS biofilm was gradually increased， and the pore sizes was ranged from 0 to 10 μm； by 26 weeks after degradation，the number of micro-porous structures was evenly distributed on rough surface of the PPC/PBS biofilm； by 4 weeks after degradation， the smooth surface of the biofilm showed exfoliation changes， but no micro-porous structures were observed； by 12 weeks after degradation， a small number of micro-porous changes were observed on the smooth surface， and the pore diameters were less than 5 μm； by 26 weeks after degradation， the number of micro-porous structures on the smooth surface was increased，and the pore diameters were less than 10 μm. Conclusion The structure of PPC/PBS biofilm is similar to that of human bone outer membrane，with a double-layer structure，good hydrophilicity，dense smooth surface，high porosity of rough surface，good biocompatibility，and slow degradation；it is an ideal guided regeneration biofilm.

Objective To discuss the effect of hydrogel-based delivery of basic fibroblast growth factor （bFGF） on the function of the NIH3T3 fibroblast cells after oxygen-glucose deprivation （OGD），and to clarify the improvement effect of bFGF-loaded hydrogel on the oxidative stress responses in the fibroblasts under the OGD condition. Methods The bFGF-containing agarose hydrogel was prepared， and the NIH3T3 cells at logarithmic growth phase were divided into blank group， 20% agarose group， and 20% agarose + glutaraldehyde group. The viabilities of the NIH3T3 cells after co-cultured with the hydrogel-eluted fluid for 6， 12， and 24 h were detected by CCK-8 assay. The NIH3T3 cells were divided into control group， OGD group， and OGD + different masses of bFGF-loaded groups （OGD + 1 ng bFGF group， OGD+10 ng bFGF group， and OGD+100 ng bFGF group）. The expression levels of α-smooth muscle actin （α-SMA）， type Ⅰ collagen， and type Ⅲ collagen proteins in the NIH3T3 cells in various groups were detected by Western blotting method； the content of malondialdehyde （MDA） and activities of lactate dehydrogenase （LDH） and superoxide dismutase （SOD） in the NIH3T3 cells were detected by assay kits； the in vitro release rate of bFGF in the NIH3T3 cells in various groups was detected by enzyme-linked immunosorbent assay （ELISA） method. Results The CCK-8 assay results showed that compared with blank group， the viability of the NIH3T3 cells between blank group， 20% agarose group， and 20% agarose+ glutaraldehyde group at different elution times had no significant difference（P>0.05）. The Western blotting results showed that the expression levels of α-SMA and type Ⅰ collagen proteins in the NIH3T3 cells in OGD group were significantly higher than that in control group （P<0.05 or P<0.01）.Compared with OGD group，the expression levels of α-SMA and type Ⅰ collagen proteins in the NIH3T3 cells in OGD+1 ng bFGF， OGD+10 ng bFGF， and OGD+100 ng bFGF groups were significantly decreased （P<0.05 or P<0.01） in a dose-dependent manner.Compared with control group，the level of MDA and activity of LDH in the NIH3T3 cells in OGD group were increased（P<0.05）， while the activity of SOD was decreased （P<0.05）. Compared with OGD group， the level of MDA and the activity of LDH in the NIH3T3 cells in OGD + 1 ng bFGF group were decreased （P<0.05）， while the activity of SOD was increased （P<0.05）.The ELISA results showed that about 10% bFGF was released from the hydrogel within 4 h， about 15% bFGF was relleased from the bydrogel with in 12 h， and about 21% bFGF was released from the hydrogel within 24 h. Conclusion The bFGF-loaded hydrogel can modulate the transformation of the fibroblasts under the OGD condition，inhibit the expression of typeⅠ collagen protein，and improve the oxidative stress responses in the fibroblasts. Moreover，this system exhibits a sustained drug-release ability.

Objective To screen out the key genes affecting lung adenocarcinoma （LUAD） through bioinformatics methods，and to analyze their biological functions and the influences on the LUAD prognosis. Methods The GSE118370 and GSE136043 chip data were downloaded from the Gene Expression Omnibus（GEO） Database. The LUAD-related data were selected from the The Cancer Genome Atlas（TCGA） Database. R software was used to analyze the co-expressed differentially expressed genes （DEGs）； clusterProfile R package was utilized for Gene Ontology （GO） functional enrichment analysis； DAVID Database was used for the Kyoto Gene and Genome Encyclopedia （KEGG） signaling pathway enrichment analysis； STRING Database was used to construct the protein-protein interaction （PPI） network； Cytoscape was used to screen out the top 10 key genes； GEPIA Database and Human Protein Atlas（HPA） Database were used to analyze the expressions of key genes mRNA and protein in normal lung tissue and LUAD tissue， and their expressions in LUAD tissues with different stages；immune infiltration analysis and survival analysis were used to analyze the correlation between the expressions of key genes and the survival time of the patients. Results In total， 428 DEGs were screened out. The GO functional analysis results showed that the DEGs of LUAD were mainly enriched in biological process（BP） such as epithelial-mesenchymal transition （EMT）， cellular component（CC） such as the cell base， and molecular function（MF） such as extracellular matrix （ECM） structure formation.The KEGG signaling pathway analysis results showed that the DEGs of LUAD were mainly enriched in the pathways like cytokine receptor interactions.topoisomerase Ⅱ alpha（TOP2A），abnormal spindle microtubule assembly（ASPM），cyclin B1，（CCNB1），cell division cycle associated 8（CDCA8），baculoviral IAP repeat containing 5（BIRC5），aurora A（AURKA），kinesin family member 20A（KIF20A），centrosomal protein 55（CEP55），centromere protein F（CENPF），and targeting protein for xklp2（TPX2） were selected as the key genes. Compared with normal lung tissue， the expression levels of TOP2A， CCNB1， CDCA8， BIRC5， AURKA， KIF20A， CEP55， CENPF， and TPX2 mRNA in lung tissue of the LUAD patients were increased （P<0.01）， and the expression levels of TOP2A， CCNB1， CDCA8， BIRC5， AURKA， KIF20A， CEP55， CENPF， and TPX2 proteins were increased. There were significant differences in the expression levels of CCNB1， CDCA8， BIRC5， AURKA， KIF20A， CEP55， and TPX2 mRNA in the LUAD tissue with different stages （P<0.01）. Compared with the LUAD patients with stage Ⅰ，stage Ⅱ，and stage Ⅲ，the expression levels of CCNB1， CDCA8， AURKA， KIF20A， CEP55， and TPX2 mRNA in lung tissue of the LUAD patients with stage Ⅳ were increased（P<0.01）； compared with the LUAD patients with stage Ⅰ，stage Ⅱ， and stage Ⅳ，the expression level of BIRC5 mRNA in lung tissue of the LUAD patients with stage Ⅲ was increased （P<0.01）. The expressions of 10 key genes were negatively correlated with the B-lymphocyte infiltration （－0.253≤r≤－0.014， P<0.01）； the expressions of TOP2A， ASPM， CDCA8， BIRC5， CEP55， CENPF， and TPX2 were positively correlated with the neutrophil infiltration （0.049≤r≤0.165，P<0.01）； the expressions of CCNB1 and AURKA were negatively correlated with the CD4 T lymphocyte， macrophage， and dendritic cell infiltration （－0.210≤r≤－0.100，P<0.01）. The high expression of CDCA8 increased the risk of LUAD deterioration （P<0.01）， and the high expressions of TOP2A， CCNB1， CDCA8， BIRC5， AURKA， KIF20A， CEP55， CENPF， and TPX2 increased the death risk of the patients（P<0.01）. Conclusion TOP2A， ASPM， CCNB1， CDCA8， BIRC5， AURKA， KIF20A， CEP55， CENPF， and TPX2 are the key genes involved in the development and progression of LUAD. They may promote the development of LUAD by accelerating the EMT process， and their high expressions suggest the poor prognosis and elevate the death risk of the LUAD patients.

Objective To discuss the risk factors of recurrence of papillary thyroid microcarcinoma （PTMC），and to provide the reference for the individualized diagnosis and treatment of PTMC. Methods The retrieval time was limited from the establishment to April 2022， and the case-control studies related to PTMC recurrence searched in PubMed， EMBase， Elsevier Science Direct and Web of Science Databases were collected.Review Manager 5.4 software was used to perform the Meta-analysis and ADDIS 1.16.8 software was used to make the Bayesian network Meta-analysis，and the risk factors of PTMC recurrence were screened out and ranked. Results Fourteen studies were included， involving 7 834 PTMC patients who underwent surgical treatment，302 patients experienced lymphnode recurrence， local thyroid bed recurrence， or distant metastasis recurrence. Age， multifocality， tumor size， extrathyroidal invasion， lymphnode metastasis， and vascular invasion of the patients were related to the PTMC recurrence （P<0.05）. Lymphnode metastasis was the most significant risk factor for the PTMC recurrence， followed by vascular invasion， and multifocality was the least significant risk factor. There were no significant correlations between gender， lesion histopathological background， gland histopathological background， radioactive iodine treatment， surgical margin， and the extent of thyroidectomy with PTMC recurrence （P>0.05）. Conclusion Lymphnode metastasis， vascular invasion， tumor size>5 mm，age，extrathyroidal invasion， and multifocality are the risk factors for the PTMC recurrence.The clinician may consider to treat the PTMC patients with routine central lymphnode dissection to prevent disease recurrence，and develop the individualized treatment plans based on the risk factor stratification.

Objective To analyze the clinical characteristics and risk factors of the patients with primary Sjogren’s syndrome （pSS） complicated with interstitial lung disease （ILD）， and to improve the clinicians’understandings of the disease， and to provide the evidence for the early detection of extradular organ involvement by the clinicians. Methods The clinical characteristics of 176 patients with pSS were collected and were divided into pSS complicated with ILD group （n=21） and pSS complicated without ILD group （n=155） according to whether the patients were complicated with ILD.The levels of hemoglobin （Hb）， white blood cells （WBC）， platelets （PLT）， C-reactive protein （CRP）， erythrocyte sedimentation rate （ESR），rheumatoid factor （RF），immunoglobulin G （IgG），immunoglobulin M （IgM），immunoglobulin A （IgA）， complement 3 （C3）， complement 4 （C4）， and the positive rates of granular type antinuclear antibodies， cytoplasmic granular type antinuclear antibodies， homogeneous type antinuclear antibodies， anti-SSA， anti-SSB， and anti-Ro-52 antibodies of the patients in two groups were compared.Univariate and multivariate Logistic regression analysis were used to analyze the clinical characteristics and laboratory indexes of the pSS patients complicated with ILD. Results Among the 176 pSS patients， the ratio of male to female was 1∶28， with the median age of 49 （39－58） years and the median course of 24 （24－48） months. Compared with pSS complicated without ILD group，the age of the patients in ILD group was older （P<0.01）， and the level of ESR was increased（P<0.01），the positive rates of cytoplasmic granular type antinuclear antibody and anti-Ro-52 antibody were increased （P<0.01）. The multivariate Logistic regression analysis results showed that the increasing of ESR level （OR=1.046，95% CI： 1.026－1.066， P=0.001） and positive granulosa type （OR=4.676，95% CI： 1.156－18.916， P=0.031） were the risk factors for ILD complicated with pSS. Conclusion The patients with pSS complicated by ILD have an older onset age and higher ESR levels； ILD is more likely to occur when the cytoplasmic granular type antinuclear antibody and anti-Ro-52 antibody of the patients are positive； the increasing of the ESR level and positive coated granular type antinuclear antibody are the risk factors for the pSS complicated with ILD.

Objective To discuss the effect of the expression of stathmin 1 （STMN1） in placenta tissue of the pre-eclampsia （PE） patients on the invasion and migration functions of the trophoblast cells， and to clarify the related mechanism. Methods The placenta tissues of 17 healthy pregnant women （control group） and 15 PE patients （PE group） were selected. The human trophoblast HTR-8 cells were divided into siRNA-STMN1 group （transfected with siRNA-STMN1）， siRNA-NC group （transfected with negative control sequence siRNA-NC），pcDNA3.1-STMN1 group （transfected with pcDNA3.1-STMN1）， and pcDNA3.1-NC group （transfected with negative control sequence pcDNA3.1-NC）. The expression intensities of STMN1 and epithelial-mesenchymal transition（EMT）-related proteins in placenta tissue of the patients in two groups were observed by immunohistochemistry （IHC） staining； the proliferation rates of the HTR-8 cells in various groups were detected by CCK-8 method； the migration abilities of the HTR-8 cells in various groups were detected by cell scratch experiment； the invasion abilities of the HTR-8 cells in various groups were detected by Transwell chamber experiment； the expression levels of STMN1 mRNA in placenta tissue of the patients in two groups and HTR-8 cells were detected by real-time fluorescence quantitative PCR （RT-qPCR） method； the expression levels of STMN1， E-cadherin， and N-cadherin proteins in placenta tissue of the patients in two groups and HTR-8 cells were detected by Western blotting method. Results Compared with control group， the systolic blood pressure， diastolic blood pressure， and the level of urine protein of the patients in PE group were significantly increased （P<0.01）， and the body weight of the newborn was increased （P<0.05）.The IHC staining results showed that compared with control group， the expression intensities of STMN1 and E-cadherin proteins in placenta tissue of the patients in PE group were increased， and the expression intensity of N-cadherin protein was decreased.The CCK-8 results showed that compared with siRNA-NC group， the proliferation rate of the HTR-8 cells in siRNA-STMN1 group was decreased（P<0.05）； compared with pcDNA3.1-NC group，the proliferation rate of the HTR-8 cells in pcDNA3.1-STMN1 group was increased（P<0.05）.The cell scratch experiment results showed that compared with siRNA-NC group，the migration rate of the HTR-8 cells in siRNA-STMN1 group was decreased（P<0.05）；compared with the pcDNA3.1-NC group， the migration rate of the HTR-8 cells in pcDNA3.1-STMN1 group was increased （P<0.05）. The Transwell chamber experiment results showed that compared with siRNA-NC group， the number of invasion cells in siRNA-STMN1 group was significantly decreased （P<0.01）； compared with pcDNA3.1-NC group， the number of invasion cells in pcDNA3.1-STMN1 group was significantly increased （P<0.01）. The RT-qPCR results showed that compared with control group， the expression level of STMN1 mRNA in placenta tissue of the patients in PE group was decreased （P<0.05）； compared with siRNA-NC group， the expression level of STMN1 mRNA in the HTR-8 cells in siRNA-STMN1 group was significantly decreased （P<0.01）； compared with pcDNA3.1-NC group， the expression level of STMN1 mRNA in the HTR-8 cells in pcDNA3.1-STMN1 group was significantly increased （P<0.01）. The Western blotting results showed that compared with control group， the expression level of STMN1 protein in placenta tissue of the patients in PE group was decreased （P<0.05）. Compared with siRNA-NC group， the expression levels of STMN1 and N-cadherin proteins in the HTR-8 cells in siRNA-STMN1 group were decreased （P<0.05）， and the expression level of E-cadherin protein was increased （P<0.05）. Compared with pcDNA3.1-NC group， the expression levels of STMN1 and N-cadherin proteins in the HTR-8 cells in pcDNA3.1-STMN1 group were increased （P<0.05）， and the expression level of E-cadherin protein was decreased （P<0.05）. Conclusion The expression of STMN1 in placenta tissue of the PE patients is significantly reduced，suggesting that STMN1 may be involved in the development of PE by regulating the EMT process of the trophoblast cells and inhibiting the cell proliferation， invasion， and migration.

Objective To discuss the expression of Klotho in placenta exosomes （Exo）of the patients with pre-eclampsia （PE），and to clarify its effect on the oxidative stress in the vascular endothelial cells. Methods The clinical data of 40 pregnant women including 20 with normal pregnancy（NP） women （NP group） and 20 PE patients （PE group） were collected. The placenta Exo in peripheral blood of the patients in two groups were isolated， and the oe-Klotho and oe-NC plasmids were transfected into the human chorionic trophoblast cells（HTR-8/SVneo）， respectively， and were regarded as oe-Klotho group and oe-NC group.The expression levels of Klotho mRNA and protein in placenta Exo and the HTR8/SVneo cells in two groups were detected by real-time fluorescence quantitative PCR （RT-qPCR） method and Western blotting method. The human umbilical vein endothelial cells （HUVECs） with good growth status were taken and divided into PE-Exo group （co-cultured with placenta Exo from the patients with PE），NP-Exo group （co-cultured with placenta Exo from the NP subjects）， oe-Klotho-Exo group （co-cultured with Exo from the HTR-8/SVneo cells transfected with oe-Klotho）， and oe-NC-Exo group （co-cultured with Exo from the HTR-8/SVneo cells transfected with oe-NC） according to the sources of Exo.The expression levels of exosomal marker proteins CD63， TSG101， and placenta Exo-specific marker PLAP protein were identified by transmission electron microscope （TEM） and Western blotting method； the levels of nitric oxide （NO）， reactive oxygen species （ROS）， and malondialdehyde （MDA） and the activities of superoxide dismutase （SOD） in the HUVECs in various groups were detected by enzyme-linked immunosorbent assay （ELISA） method；the expression levels of endothelial nitric oxide synthase （eNOS） mRNA in the HUVECs in various groups was detected by RT-qPCR method；the expression levels of eNOS protein in the HUVECs in various groups were detected by Western blotting method. Results Compared with NP group， the systolic and diastolic blood pressures of the patients in PE group were increased （P<0.05）， the body weight of newborn and placenta weight were significantly decreased （P<0.05 or P<0.01）. The TEM observation results showed that the placenta Exo derived from the subjects in both NP and PE groups being round or oval vesicular discoid structures with complete membrane and similar shape， with a diameter of 50—100 nm. The Exo from the subjects in both groups highly expressed Exo marker proteins CD63 and TSG101， and placental Exo-specific protein PLAP. The nanoparticle tracking analysis （NTA） results showed that the particle size of Exo was 50—200 nm， suggesting that the vesicles isolated from the peripheral blood were derived from placental Exo. The TEM， Western blotting， and NTA results confirmed that the vesicles from oe-NC group and oe-klotho group had typical Exo structures， molecular markers， and particle size characteristics. The Exo with green fluorescence expression could be observed in the HUVECs， suggesting that the placenta Exo could be taken up by the HUVECs. The ELISA results showed that compared with NP-Exo group， the level of NO and activity of SOD in PE-Exo group were significantly decreasd（P<0.05 or P<0.01）， and the levels of ROS and MDA were significantly increased（P<0.05 or P<0.01）； compared with oe-NC-Exo group， the level of NO and activity of SOD in the HUVECs in oe-Klotho-Exo group were increased （P<0.05）， and the levels of ROS and MDA were significantly decreased （P<0.05 or P<0.01）. The RT-qPCR results showed that compared with NP group， the expression level of Klotho mRNA in placenta Exo of the patients in PE group was significantly decreased （P<0.01），and the expression level of eNOS mRNA in the HUVECs was significantly decreased （P<0.01）； compared with oe-NC group， the expression level of Klotho mRNA in Exo in the trophoblast cells in oe-Klotho group was significantly increased （P<0.01）； compared with oe-NC-Exo group， the expression level of eNOS mRNA in the HUVECs in oe-Klotho-Exo group was significantly increased （P<0.01）. The Western blotting results showed that compared with NP group， the expression level of Klotho protein in placenta Exo of the patients in PE group was significantly decreased （P<0.01）； compared with NP-Exo group， the expression level of eNOS protein in the HUVECs in PE-Exo group was significantly decreased （P<0.01）； compared with oe-NC group， the expression level of Klotho protein in Exo in the trophoblast cells in oe-Klotho group was significantly increased （P<0.01）； compared with oe-NC-Exo group， the expression level of eNOS protein in the HUVECs in oe-Klotho-Exo group was significantly increased （P<0.01）. Conclusion The placenta Exo from the PE patients may inhibit the production of NO in the HUVECs and promote the oxidative stress to impair the endothelial cell function. The over-expression of Klotho in Exo in the trophoblast cells can reduce the production of NO and level of oxidative stress in the HUVECs.

Objective To discuss the expression levels of cyclooxygenase-2 （COX-2） and β-catenin in the in situ endometrium tissue， lesion tissue， and serum of the adenomyosis （AM） patients，and to clarify their relationships with dysmenorrhea. Methods A total of 90 cases of confirmed AM patients with dysmenorrhea who underwent abdominal hysterectomy were selected as AM group， and 30 cases of patients with uterine myomas （UM） without dysmenorrhea were selected as UM group. The degree of dysmenorrhea of the AM patients was assessed by using the visual analogue scale （VAS）， and 90 cases of AM patients were divided into mild dysmenorrhea group， moderate dysmenorrhea group， and severe dysmenorrhea group （n=30）. Immunohistochemistry （IHC） was used to detect the expression levels of COX-2 and β-catenin proteins in endometrium tissue of the patients in two groups； enzyme-linked immunosorbent assay （ELISA） method was used to detect the levels of COX-2 and β-catenin in serum of the patients in two groups；the association between the expression levels of COX-2 and β-catenin in endometrium tissue of the patients in two groups and degree of dysmenorrhea of the patients was analyzed by Spearman correlation analysis. Results The COX-2 protein expression was mainly located in the cytoplasm of endometrium tissue， while β-catenin protein was mainly expressed in the cell membrane or cytoplasm of endometrium tissue. Compared with UM group， the expression levels of COX-2 protein in in situ endometrium tissue and lesion tissue of the patients in AM group were increased（P<0.05），and the expression level of β-catenin protein was decreased（P<0.05）. Compared with in situ endometrium tissue of the patients in AM group， the expression level of COX-2 protein in lesion endometrium tissue of the patients in AM group was increased（P<0.05）.The expression level of COX-2 protein in the lesion tissue of the AM patients with moderate dysmenorrhea and the expression level of COX-2 protein in in situ endometrium and lesion tissues of the AM patients in severe dysmenorrhea group were significantly higher than those in mild dysmenorrhea group（P<0.05）； the expression level of COX-2 protein in in situ endometrium and lesion tissues of the AM patients in severe dysmenorrhea group were significantly higher than those in moderate dysmenorrhea group（P<0.05）.There were no significant differences in the expression levels of β-catenin protein in in situ endometrium tissue and lesion tissue of the AM patients between different degrees of dysmenorrhea groups （P>0.05）. Compared with UM group，the serum level of COX-2 of the patients in AM group was significantly increased （P<0.05），and the serum levels of β-catenin of the patients in AM group was decreased （P<0.05）.Compared with mild dysmenorrhea group，the serum COX-2 levels of the patients in moderate and severe dysmenorrhea groups were increased（P<0.05）. There were no significant differences in serum β-catenin level of the patients between different degrees of dysmenorrhea groups （P>0.05）.The expression level of COX-2 protein in lesion tissue of the AM patients was negatively correlated with the expression level of β-catenin protein （r=－0.364， P<0.05）； in in situ endometrium and lesion tissues of the AM patients， the expression level of COX-2 protein was positively correlated with the degree of dysmenorrhea （r=0.511，P<0.05； r=0.696， P<0.05）， suggesting that the expression level of COX-2 protein was positively correlated with the degree of dysmenorrhea and there was no correlation between the expression level of β-catenin protein and the degree of dysmenorrhea （P>0.05）. The correlation between the serum levels of COX-2 and β-catenin of the patients were negative （r=－0.534， P<0.05）， and the COX-2 level of the patients was positively correlated with the degree of dysmenorrhea （r=0.613， P<0.05）. Conclusion The increasing of expression level of COX-2 and decreasing of expression level of β-catenin and their correlation both induce AM development and endometriotic invasion， and high expression of COX-2 may promote the dysmenorrhea， which may serve as a potential molecular target for the treatment of AM and associated dysmenorrhea.

Objective To discuss the pregnancy outcomes of the patients with different body mass index （BMI） underwent fresh embryo transfer cycles and frozen-thawed embryo transfer （FET） cycles after treated with long regimen controlled ovarian hyperstimulation （COH）， and to analyze the correlation between BMI and the pregnancy outcomes after treated with assisted reproductive technology （ART）. Methods The patients who underwent long regimen COH were divided into fresh embryo transfer cycle group （n=1 957） and FET cycle group （n=2 328）， according to their BMI， the patients in both groups were further classified into lean group（BMI <18.5 kg·m^－2）， normal group（18.5 kg·m^－2 ≤BMI <24.0 kg·m^－2）， overweight group（24.0 kg·m^－2 ≤BMI <28.0 kg·m^－2）， and obese group（BMI≥28.0 kg·m^－2）.The general informations， COH conditions， embryo conditions and clinical pregnancy outcomes of the patients in various groups were collected and analyzed. Results There were 1 957 patients， 1 957 cycles in fresh embryo transfer cycle group， which included 103 cycles in lean group， 1 254 cycles in normal group， 476 cycles in overweight group and 124 cycles in obese group. Compared with normal group， the levels of basal follicle-stimulating hormone （bFSH）， basal estradiol （bE2） and basal luteinizing hormone （bLH） of the patients in lean group were significantly increased （P<0.01）； the age of the patients in overweight group was increased （P<0.05）， the levels of bFSH， bE2， basal progesterone （bP） and bLH of the patients in overweight group were significantly decreased （P<0.01）； the levels of bFSH， bE2， and bLH of the patients in obese group were significantly decreased （P<0.01）， while the infertility time and basal testosterone （bT） level were significantly increased （P<0.05 or P<0.01）. Compared with lean group， the use time of gonadotropins（Gn） of the patients in overweight group was significantly increased （P<0.01）， the fertilization rates and transferrable embryo rates of the patients in normal， overweight and obese groups were decreased （P<0.01）. In FET cycle group， there were 1 914 patients， 2 328 cycles， including 128 cycles in lean group， 1 503 cycles in normal group， 557 cycles in overweight group and 140 cycles in obese group. Compared with lean group， the ages of patients in normal， overweight and obese groups were significantly increased （P<0.05 or P<0.01）. Conclusion For the infertile patients who are going to undergo the in vitro fertilization （IVF）， maintaining normal body weight could reduce the dosage of ovulation induction drugs， obtain more embryos， and offer more chances of pregnancy.

Objective To discuss the change of reticulocyte hemoglobin （RET-He） level of the anemic patients with different degrees and different types of anemia， and to analyze its auxiliary diagnostic value in microcytic hypochromic anemia. Methods The clinical data of 369 anemic patients and 40 healthy physical examination individuals were collected. The patients were divided into mild anemia group （125 cases）， moderate anemia group （163 cases）， and severe anemia group （81 cases） based on the hemoglobin （Hb） level，and 40 healthy physical examination individuals were regarded as control group. The patients were also divided into macrocytic anemia group （91 cases）， normocytic anemia group （108 cases）， simple microcytic anemia group （23 cases）， and microcytic hypochromic anemia group （147 cases） based on the morphological types of anemia. The red blood cell count， Hb level， blood cell specific volume （HCT）， mean red blood cell volume （MCV）， mean red blood cell Hb level （MCH），mean red blood cell Hb concentration （MCHC）， and RET-He level of the subjects in various groups were detected；the receiver operating characteristic （ROC） curve was drawn to obtain the diagnostic cutoff value for the disease； the area under curve （AUC）， sensitivity， and specificity were calculated. Results Compared with control group， the RET-He levels of the patients in moderate and severe anemia groups were significantly decreased （P<0.01）； compared with mild anemia group， the RET-He levels of the patients in moderate and severe anemia groups were significantly decreased（P<0.01）. The RET-He levels of the patients in macrocytic anemia， normocytic anemia， simple microcytic anemia， and microcytic hypochromic anemia groups showed a decreasing trend （P<0.01）； compared with control group， the RET-He levels of the patients in simple microcytic anemia and microcytic hypochromic anemia groups were significantly decreased （P<0.01）； compared with mild macrocytic anemia group， the RET-He levels of the patients in mild normocytic anemia， mild simple microcytic anemia， and mild microcytic hypochromic anemia groups were significantly decreased （P<0.01）. Compared with mild normocytic anemia group， the RET-He levels of the patients in mild simple microcytic anemia group and mild microcytic hypochromic anemia group were significantly decreased （P<0.01）. Compared with moderate macrocytic anemia group， the RET-He levels of the patients in moderate normocytic anemia group， moderate microcytic anemia group， and moderate microcytic hypochromic anemia group were significantly decreased （P<0.05 or P<0.01）；compared with moderate normocytic anemia group， the RET-He level of the patients in moderate microcytic hypochromic anemia group was significantly decreased （P<0.01）； compared with severe macrocytic anemia group and severe normocytic anemia group， the RET-He level of the patients in severe microcytic hypochromic anemia group was significantly decreased （P<0.01）；compared with moderate simple microcytic anemia group， the RET-He level of the patients in severe simple microcytic anemia group was significantly increased （P<0.01）； compared with mild microcytic hypochromic anemia group， the RET-He levels of the patients in moderate microcytic hypochromic anemia group and severe microcytic hypochromic anemia group were significantly decreased （P<0.01）；compared with moderate microcytic hypochromic anemia group， the RET-He level of the patients in severe microcytic hypochromic anemia was significantly decreased （P<0.01）.The cutoff value of RET-He level for diagnosing microcytic hypochromic anemia was 27.25 pg， and the sensitivity was 91.2%， the specificity was 90.5%， and the AUC was 0.968［95% confidence interval （CI）：0.952—0.984］， suggesting that the RET-He level could make an auxiliary diagnosis for microcytic hypochromic anemia. Conclusion The RET-He level can assist in the diagnosis of anemia with different morphological types and degrees， and has certain clinical application value for the diagnosis of microcytic hypochromic anemia.

Objective To discuss the influence factors of retromolar space in the patients with different osteofacial types， and to analyze the correlation between the mandibular bone structure and retromolar space and craniofacial structure. Methods A total of 200 adult orthodontic patients with fully erupted dentition except the third molars and crowding less than 5 mm were chosen and divided into skeletal type Ⅰ low angle group， skeletal type Ⅰ average angle group， skeletal type Ⅰ high angle group， skeletal type Ⅱ average angle group， and skeletal type Ⅲ average angle group. The sella nasion A point angle（SNA）， sella nasion B point angle（SNB）， AB plane angle（ANB）， skull base anterior-mandibular plane angle（SN-MP），facial height index（FHI），mandibular ramus length（Go-Co），and mandibular body length（Go-Gn） of all the patients were detected. Cone-beam computed tomography （CBCT） technology was used to detect the shortest distances C0 and C2 from the mandibular second molar to the anterior border of the ramus at the planes parallel to the cuspid line with a depth of 0 and 2 mm form the occlusal plane， and the shortest distances R2， R4， R6， R8， and R10 from the tooth root to the lingual cortical bone of the mandible at the enamel-bone junction in the root direction 2， 4， 6， 8， and 10 mm planes.The correlation was analyzed by using Spearman correlation analysis. Results Compared with skeletal type Ⅰ low angle group， the SN-MP of the patients in skeletal type Ⅰ average angle group and skeletal type Ⅰ high angle group was increased significantly （P<0.01）， and the FHI was decreased significantly （P<0.01）. Compared with skeletal type Ⅰ average angle group， the SN-MP of the patients in skeletal type I high angle group was increased significantly （P<0.01），FHI was decreased significantly （P<0.01）. Compared with skeletal type Ⅰ average angle group， the ANB of patients in skeletal type Ⅱaverage angle group was significantly increased （P<0.01）， while the ANB of the patients in skeletal type Ⅲ average angle group were significantly decreased （P<0.01）. Compared with skeletal type Ⅱ average angle group， the ANB of the patients in skeletal type Ⅲ average angle group was significantly decreased （P<0.01）. Under the C2， R2， R4， R6， R8， R10 planes and in the Go-Co and Go-Gn， compared with skeletal type Ⅰ low angle group， the retromolar spaces of the patients in skeletal type Ⅰ average angle group and the skeletal type Ⅰ high angle group were significantly decreased （P<0.01）； compared with skeletal type Ⅰ average angle group， the retromolar space of the patients in skeletal type Ⅰ high angle group was significantly decreased （P<0.01）. Under the C0 plane， compared with skeletal type Ⅰ low angle group and the skeletal type Ⅰ average angle group， the retromolar space of the patients in skeletal type Ⅰ high angle group was decreased （P<0.01）. Under the C0， R2， R4， R6， R8， and R10 planes， compared with skeletal type Ⅰ average angle group， the retromolar space of the patients in skeletal type Ⅱ average angle group was significantly decreased （P<0.01）， the retromolar space of the patients in skeletal type Ⅲ average angle group was increased （P<0.01）； compared with skeletal type Ⅱ average angle group， the retromolar space of the patients in skeletal type Ⅲ average angle group was increased （P<0.01）.Under the C2 plane and in the Go-Co and Go-Gn， compared with skeletal type Ⅰ average angle group and skeletal type Ⅱ average angle group， the retromolar space of the patients in skeletal type Ⅲ average angle group was increased （P<0.01）. The retromolar space，Go-Co and Go-Gn of the patients in skeletal type Ⅰ low angle group， skeletal type Ⅰ average angle group， and skeletal type Ⅰ high angle group under all planes had positive correlations with the SNB and FHI （P<0.01）， and a negative correlation with SN-MP （P<0.01）；the retromolar space at all levels had a positive correlation with Go-Co （P<0.01）； except for the C2 plane， the remaining retromolar space under all planes had positive correlations with Go-Gn （P<0.01）；there was a positive correlation between Go-Co and Go-Gn （P<0.01）；there was a negative correlation between Go-Gn and ANB（（P<0.01）.The retromolar space and Go-Co and Go-Gn of the patients in skeletal type Ⅰ average angle group， skeletal type Ⅱ average angle group， and skeletal type Ⅲ average angle group under all planes had positive correlations with SNB （P<0.01），and the retromolar space had positive correlations with Go-Co and Go-Gn （P<0.01）； there was a positive correlation between Go-Co and Go-Gn （P<0.01）；there was a positive correlation between Go-Co and FHI（P<0.01）. Conclusion The skeletal type is an important influence factor of the retromolar space of mandible， when the molar distalization plan is formulated， the skeletal type of the patient should be considered； CBCT should be used to evaluate the available space for mandibular molar distalization.

Objective To discuss the risk factors of renal function preservation and trifecta of the patients with renal cell carcinoma（RCC） after robot-assisted partial nephrectomy （RAPN）， and to provide the evidences for the preperative evaluation，postoperative treatment and long-term follow-up. Methods A retrospective analysis was conducted on the the clinical data of 111 cases of RCC patients undergoing RAPN.The patients were divided into trifecta group （n=73） and non-trifecta group（n=38） according to whether the trifecta outcome was achieved； according to the changes of 24 h etimated glomerular fltration rates（eGFR） before and after operation，the patients were divided into postoperative 24 h eGFR decreasing≤10% group（n=85） and postoperative 24 h eGFR decreasing>10% group（n=26）.The age， gender，American Society of Anesthesiologists（ASA） score， body mass index （BMI）， hypertension，diabetes，preoperative eGFR，percentages of postoperative 24 h eGFR change， hilar tumor，dorsal ventral positions of tumor，maximum tumor diameters， sugrical aproaches，warm ischemia time（WIT），intraoperative estimated blood loss （EBL），tumor pathological types，tumor TNM stages， RENAL scores， PADUA scores， central indexes （C-index）， renal tumor invasion indexes （RTII），and tumor contact area （CSA）of the patients in two groups were compared.Multivariate Logistic regression was used to analyze the influencing factors of the patients’ achievement of trifecta and the postoperative 24 h eGFR decreasing>10%. Multiple linear regression was used to analyze the influencing factors of the changes of postoperative 24 h eGFR. Results A total of 73 patients achieved trifecta among all the 111 patients.The univariate analysis results showed that there were statistically significant differences in age， hypertension， tumor maximum diameter， RENAL score， PADUA score， C-index， RTII， CSA，and EBL of the patients in trifecta group and non-trifecta group. The multivariate Logistic analysis results showed that EBL was an independent influencing factor for failing to achieve trifecta of the patients after RAPN（OR=1.006，95%CI=1.001—1.011，P=0.020）.There were statistically significant differences in tumor maximum diameters， RENAL scores， PADUA scores， C-indexes， RTII， CSA， WIT， EBL， and tumor TNM stages of the patients（P<0.05） in 24 h postoperative eGFR decreasing > 10% and postoperative 24 h eGFR decreaing ≤10% groups. The multivariate Logisitc regression analysis results showed that RTII was an independent influencing factor for the postoperative 24 h eGFR decreasing> 10% of the patients （OR=4.442，95%CI=1.049—18.806，P=0.043）.The tumor maximum diameter，RENAL，PADUA，C-index， RTII， CSA， WIT， EBL，and tumor TNM stage had no correlation with the postoperative eGFR changes； RTII had negative correlationship with postoperative 24 h eGFR changes（B=－7.204，95%CI=－14.305－－0.102，P=0.047）. Conclusion EBL is an independent influencing factor for failure to achieve trifecta outcomes of the patients after RAPN； RTII is negatively correlated with the postoperative 24 h eGFR changes.

objective To discuss the morphological changes and related factors of soft and bone tissues in chin in the skeletal class Ⅲ malocclusion patients at three dimensions after orthognathic operation. Methods A total of 19 adult patients underwent orthognathic operation were collected， including 9 males and 10 females. All the patients received craniofacial computed tomography （CT） scans one week before operation（T0） and 12 months after （T1） operation to obtain the facial data of the soft and bone tissues， with a three-dimensional reconstruction and registration by using ProPlan CMF 3.0 software. The measurements and calculations were made on the thickness of chin soft tissue， the horizontal， sagittal， and vertical changes in anatomical landmarks， and the correlation and ratios between them. Results The most significant retrusion of soft tissue after operation was in the lower lip vermilion and the mentolabial fold area. The posterior displacement of soft tissue was gradually decreased， spreading from this area to the periphery. The posterior change in the submental area was relatively minor， with an anterior trend at the submental point. Compared with T0 time point， the soft tissue thickness at the lower lip （LL）at T1 time point was increased （P<0.05）.The linear regression analysis results showed that at the horizontal（X）， as the bone tissue changed to the left，the anatomical landmarks of soft tissue in chin LL （B=0.795， R2=0.832）， mentolabial sulcus （Si） （B=0.876，R2=0.987）， soft tissue pogonion （Pos） （B=0.890， R2=0.971）， and menton of soft tissue（Mes） （B=0.942，R2=0.978） showed negative changes， all moved to the left； the movement directions of soft and bone tissues were consistent. At the sagittal （Y） direction， as the bone tissue moved backward， the LL （B=0.882， R2=0.934）， Si （B=0.946， R2=0.847）， Pos （B=0.839， R2=0.909） and Mes （B=0.666，R2=0.455） all moved backward，and the movement directions of soft and bone tissues were consistent.At the vertical （Z） direction， as the bone tissue moved upwards， the LL （B=0.932， R2=0.686）， Si （B=0.834， R2=0.469）， and Mes （B=0.925， R2=0.709） moved downwards， while the Pos （B=0.487， R2=0.444） moved upwards. In horizontal， sagittal， and vertical directions， the correlation between soft-tissue movement（ΔSM） and bone-tissue movement（ΔBM） of chin anatomical landmarks was generally strong （0.6<r<1.0，P<0.01）. Conclusion The changes of soft and hard tissues in the chin of skeletal class Ⅲ malocclusion patients at horizontal， sagittal， and vertical directions after orthognathic operation are influenced by bone tissue movements， showing the positive correlation. The correlations at the horizontal and sagittal directions are stronger， and the correlation at the vertical direction is weaker.

Objective To discuss the effect factors of enteral nutrition feeding intolerance （FI） of the patients with severe acute pancreatitis （SAP）， and to construct and verify the risk prediction model. Methods The data of 246 SAP patients were collected and the patients were divided into tolerance group （n=143） and non-tolerance group （n=103） according to whether the enteral nutrition FI occurred.Age， intra-abdominal pressure， hypertriglyceridemia， hypoproteinemia， and other related data of the patients in two groups were compared， the predictive factors of FI were screened out by Logistic regression analysis，and the prediction model was constructed.The area under receiver operating characteristic （ROC） curve （AUC） and Hosmer-Lemeshow test were used to verify the prediction effect of the model. Ninety-two SAP patients were selected to verify the predictive effect of the model. Results A total of 6 predictors，such as hypertriglyceridemia （OR=1.962， 95%CI：1.088－3.538，P=0.025），hypoproteinemia（OR=1.920， 95%CI：1.049－3.516，P=0.034），acute physiological and chronic health status Ⅱ（APACHEⅡ）≥score 20 points （OR=3.118， 95%CI：1.720－5.653，P<0.01），intra-abdominal pressure ≥12 mmHg （OR=2.826， 95%CI：1.534－5.205，P=0.001），time to start enteral nutrition ≥72 h（OR=2.350，95%CI：1.179－4.683，P=0.015）， and addition of microecological agent （OR=0.379， 95%CI：0.209－0.685，P=0.001） were included. The formula of the model was Z=－1.206+0.674×hypertriglyceridemia+0.652×number of patients with hypoproteinemia+1.137×number of patients with APACHEⅡ score ≥20 points +1.039×number of patients with intra-abdominal pressure ≥12 mmHg+0.855×number of patients with time to start enteral nutrition ≥72 h－0.971×number of patients with　addition of microecological agent. The Hosmer-Lemeshow test results showed that χ²=5.985 and P=0.901， and the AUC of the prediction model was 0.793 （95%CI： 0.735－0.851， P<0.01），the Yoden index was 0.498，the optimal critical value was 0.499， the sensitivity was 0.818， and the specificity was 0.680. The agent verification results of the model showed that the AUC was 0.880 （95%CI： 0.811－0.948，P<0.01），the sensitivity was 0.774，the specificity was 0.867， and the accuracy rate was 81.52%. Conclusion The constructed risk prediction model of SAP enteral nutrition FI has high sensitivity and specificity， and good prediction efficiency.

Objective To analyze the imaging performance and clinical diagnosis and treatment process of one patient with peri-gallbladder fluid caused by the posterior duodenal perforation primarily diagnosed by ultrasound， and to provide the clinical diagnostic evidence for this disease. Methods The clinical data， laboratory examination， gastroscope performance， and imaging performance of one patient with peri-gallbladder fluid caused by the posterior duodenal perforation was collected. The process of diagnosis and treatment was recorded and followed up. The related literatures were reviewed to analyze the clinical characteristics and imaging performances of the posterior duodenal perforation. Results The patient was a 50-year-old male who had constant dull pain in the upper right abdomen for over 20 d， without obvious cause and intensified after meals， accompanied by radiculalgia in the right waist and back.The ultrasonic examination at a local hospital showed an occupying lesion in the gallbladder， and the patient came to our hospital for the further diagnosis and treatment. On the day of admission， the ultrasound examination showed poor fasting gallbladder filling， continuous and uniformly thickened gallbladder wall.The multiple strong echoes were observed in the gallbladder cavity， and the chaotic distributed hypoechoic and anechoic areas could be seen around the gallbladder， extending to the back of the duodenal bulb， and connected with the bulb by a narrow strip of gaseous shadow. The ultrasound results showed that there was perforation of the posterior wall of the duodenal bulb， leading to pericholecystic fluid accumulation， with poor gallbladder filling due to compression， and chronic inflammation of the gallbladder wall associated with gallstones. The enhanced computed tomography （CT） results clearly showed the perforation site in the posterior wall of the duodenal bulb， and the CT conclusion was consistent with the ultrasound conclusion. The gastroscope results showed a large deep ulcer on the posterior wall of the duodenal bulb， and it confirmed to be diagnosed as the peri-gallbladder fluid caused by ulcer perforation. A gastric tube and jejunal nutrition tube were inserted， and the symptomatic treatments such as anti-inflammation， analgesia， and acid suppression were carried out. The ultrasound reexamination within 3 months of treatment showed the peri-gallbladder fluid was gradually decreased and the gallbladder cavity was gradually filled.Since the patient’s gallbladder inflammation symptoms persisted，the cholecystectomy was performed， and it was pathologically diagnosed as chronic cholecystitis complicated with gallstones. Conclusion The posterior duodenal perforation is often misdiagnosed or missed due to atypical clinical manifestations.The imaging examination can assist in early clinical diagnosis. When the presence of abdominal gas does not interfere with observation， the ultrasound can serve as the first choice and effective method for the diagnosis of gastrointestinal perforation.

Objective To discuss the safety of secukinumab treatment during pregnancy，and to provide the reference for the treatment of psoriasis patients during pregnancy. Methods A case of pustular psoriasis safely treated with secukinumab during pregnancy was collected and combined with the relevant literature review， the safety of secukinumab treatment for the psoriasis during pregnancy was analyzed. Results The patient was a 28-year-old woman with a body weight of 50 kg. The patient had been experiencing generalized erythema and scales for 5 years， which worsened with pustules for 1 year.The dermatology examination results showed large patches of erythema， silver-white scales and small pustules on the skin of the patient， no changes in the nails， and was not accompanied by the joint symptoms. The patient was diagnosed as generalized pustular psoriasis. After being treated with secukinumab for 2 months， the psoriasis area and severity index （PASI） was 0； the patient unexpectedly became pregnant after treated for 10 months.Secukinumab was continued throughout the pregnancy until 20 d before delivery， and the patient gave birth to a healthy full-term female baby with the body weight of 3.6 kg. Conclusion Secukinumab can be used as a therapeutic option for the patients with psoriasis during pregnancy， but more clinical cases and drug research are needed to evaluate its safety during pregnancy.

Objective： To analyze the clinical performance and diagnosis process of the autoimmune encephalitis （AE） patients with positive double antibodies in the cerebrospinal fluid（CSF）， and to provide the references for the diagnosis and treatment of such patients. Methods The clinical manifestations， magnetic resonance imaging （MRI） of the head， electroencephalogram （EEG）， CSF characteristics， and prognosis of one patient with AE positive for anti-glutamic acid decarboxylase （GAD） 65 and anti-γ-aminobutyric acid B receptor （GABA_BR） double antibodies in the CSF were retrospectively analyzed，and the literatures were reviewed. Results The patient was a 47-year-old male with subacute onset and progressively aggravated symptoms， mainly presenting with headaches and episodic convulsions， and blurred consciousness.The MRI results of the head suggested that the lesions were located on the right side of the cerebral falx in the frontal and parieto-occipital lobes；the CSF was positive for the anti-GAD65 and anti-GABA_BR double antibodies， and the EEG results showed the abnormal spike and slow waves， so the patient was diagnosed as AE. After anti-inflammatory and other symptomatic treatments， the patient was gradually improved and was discharged；the patient was given continuous oral corticosteroid treatment， but after 5 months， the patient was relapsed with acute onset， presenting with convulsion accompanied by drooling from the corner of the mouth. The head MRI results showed there was an abnormal high signal in the right temporal lobe. After corticosteroid treatment， the patient was improved. Conclusion The AE patients with positive double antibodies in CSF are more likely to relapse. Steroid anti-inflammatory treatment is effective. When intracranial lesions are located in the frontal and parieto-occipital lobes， it should be considered the possibility of symptoms such as convulsions. It is necessary to complete the EEG and the other inspections as soon as possible.

Objective To analyze the clinical data of the patients with non-syndromic supernumerary teeth （NSMST）， and to explore the possible mechanism of occurrence of supernumerary teeth， diagnostic criteria，and treatment method， and to enhance the comprehension of the clinicians about this disease. Methods The clinical data of one patient with NSMST were collected. Based on the specialist examination and relevant literature review results， the causes， diagnostic points and treatment principles of the supernumerary teeth were analysed. Results The patient， a 19-year-old male， checked in the clinic due to his subjective feeling of facial asymmetry， denying any systemic disease or history of genetic disorders. The intraoral examination results showed 4 supernumerary teeth， all of them showed the resembling molars morphology. The cone beam CT （CBCT） results showed that the roots of 4 erupted supernumerary teeth were fully developed. One impacted supernumerary tooth could be seen on the palatal root side of tooth 18， resembling a premolar， with fully developed roots， and there was a calcified crown shadow being observed distal mesially， and no root formation could be seen. The X-ray cephalometric analysis results showed a smaller SNB angle than the normal value， indicating the deficiency in the mandibular development； a larger FH-MP angle indicated a steep mandibular plane and high-angle pattern. There were a total of 6 supernumerary teeth on the right maxillary molar area， so the diagnosis was NSMST. The surgical extraction of the supernumerary teeth was recommended， followed by combined orthognathic-orthodontic treatment to restore normal facial morphology and dental arch form. The patient did not undergo treatment due to his personal reasons. Conclusion The NSMST patients needs to be definitely diagnosed through the imaging examinations clinically，the surgical removal is the principle treatment method，and early discovery and treatment can minimise the impact on the patient’s facial region and dentition.

Objective To discuss the influencing factors of individualized surgical treatment for the severe cicatricial ectropion， and to provide the reference for the personalized clinical treatment of such patients. Methods The clinical data of one patient who suffered from severe ectropion of both upper and lower eyelids for 29 years and exposure keratitis after burn injury were reported. The treatment included horizontal shortening of the eyelid posterior layer and full-thickness skin grafting. The diagnosis and treatment processes of the patient was analyzed， and the literatures were reviewed. Results The female patient， 33 years old， was admitted to our hospital for “burn-induced ectropion of both upper and lower eyelids for 29 years， aggravated in the past year”. The ophthalmic examination results showed significant loss of anterior skin and orbicularis oculi muscle and horizontal elongation of the posterior layer of the eyelid and conjunctiva， diagnosed as cicatricial ectropion and exposure keratitis. The operation avoided the connection of upper and lower eyelid incisions， extended the range of scar loosening to the medial side near the nasal root beyond the inner canthus， the lateral side beyond the outer canthus， and reached the orbital rim on the upper and lower sides. The horizontal shortening of the eyelid posterior layer was performed， and four full-thickness skin grafts were used to repair the anterior layer defects of the upper and lower eyelids. On the 14th day after operation， the grafts showed normal skin color， good eyelid morphology， and tight adhesion to the ocular surface； in the 12th month after operation， the grafts were completely alive， without pigmentation deposition； the eyelid morphology was normal，and there were no ectropion and good mobility； the corneal ulcer was healed， the visual acuity was improved， and the patient was satisfied with the postoperative appearance. Conclusion For the patients with severe cicatricial ectropion， completing the loosening of scar traction， maximum preservation and reinforcement of orbicularis oculi muscle， selective excision of tarsal plate， and determination of flap or graft size and thickness are the essential factors in the personalized treatment. Clinically， the conditions of the patient should be comprehensive assessmented combined with the surgical procedure selection and surgical details，which is needed to achieve the desired surgical outcomes.

Objective To analyze the clinical presentation， pathogenesis， diagnosis， and treatment of one patient with Wilson’s disease （WD），who presenting the psychiatric symptoms as the first manifestation， and to enhance the clinician’s understandings of WD. Methods The clinical data of one WD patient with obvious neuropsychiatric manifestation were collected， and the clinical characteristics and methods of diagnosis and treatment were analyzed， and the literatures were reviewed. Results The patient， a 47-year-old male， first presented tremors 9 years ago， and was diagnosed as decompensated cirrhosis， likely caused by ethanol； eight years ago， the patient developed delusions and fear， and was diagnosed as schizophrenia. Upon admission， the patient had the facial features of chronic liver disease， clear consciousness， postural and resting tremors in his limbs， increased muscular tone， bradykinesia， and dysphasia. The examination results showed low ceruloplasmin and serum copper levels， and the level of 24 h urine copper was high， the Kayser-Fleischer （K-F） ring was positive， and two point mutations were identified in the 8th exon of ATP7B gene， leading to a diagnosis of WD based on the Leipzig scoring system （score of 8）. The further magnetic resonance imaging（MRI） examination results showed abnormal signals in bilateral basal ganglia， midbrain and pons， which was consistent with the WD presentation. After treated with trientine and zinc to chelate copper， the level of urine copper of the patient was initially increased and then gradually decreased. After three cycles of copper chelation therapy， the patient’s neurological symptoms was improved. The tremors of the patient was slightly improved，and the patient could have short verbal communication with the family members， and was able to walk for short distances， which demonstrated the effectiveness of the treatment. Conclusion For the WD patients with psychiatric disorders as the first manifestation and combined liver involvement， the clinicians should pay special attention to the differential diagnosis，complete ceruloplasmin，K-F ring ophthalmology， and head MRI examinations as early as possible， and if necessary， the gene detection and（or） liver biopsy should be completed. Once the patient was diagnosed as WD，the copper chelation treatment should be initiated as soon as possible to reduce the misdiagnosis or missed diagnosis.

Objective To discuss the effect of shear wave elastography （SWE） on the differential diagnosis of benign and malignant breast non-mass lesion （NML）， and to clarify its clinical significance. Methods A total of 158 patients with breast NML were selected and divided into benign lesion group （ n=83） and malignant lesion group（ n=75） according to the postoperative pathological type of the patients.The routine ultrasound and SWE examinations were conducted before operation， and the SWE parameters of the lesions， including the maximum （E_max）， average （E_mean），minimum （E_min）， standard deviation （E_sd） and whether the stiff rim sign presented or not at 1， 2 and 3 mm around the lesion were recorded. The SWE characteristics and differences in SWE parameters of the NML patients between two groups were analyzed， and the receiver operating characteristic curve（ROC） was plotted， and the area under curve （AUC）， specificity and sensitivity were calculated. Results There were statistically significant differences in age， lesion size， palpation， and whether menopause had crested of the NML patients between two groups（ P<0.01）. All the lesions of the patients in two groups appeared as hyperechoic and irregular in shape. Compared with benign lesion group， the percentage of the patients with non-parallel， calcification and posterior echo attenuation in malignant lesion group was significantly increased （ P<0.01）， and the percentage of the patients without blood supply was significantly decreased （ P<0.01）. Compared with benign lesion group， the E_max， E_mean， and E_sd of the NML patients in malignant lesion group were significantly increased （ P<0.01）， and the E_max， E_mean， and E_sd at 1， 2， and 3 mm around the lesion were all increased （ P<0.01）. The stiff rim sign manifested as a circular or semi-circular red region around the lesions of the patients in malignant lesion group. Compared with benign lesion group， the percentage of hard rim sign of the NML patients in malignant lesion group was significantly increased （ P<0.01）.Compared with inside the lesion，the E_min at 1，2 and 3 mm around the lesion of the NML patients in benign lesion group and malignant lesion group were decreased（ P<0.01）. The AUC of E_max at 1， 2 and 3 mm around the lesion were 0.872， 0.860， 0.873 and 0.866，respectively； the AUC of E_mean were 0.796， 0.822， 0.820 and 0.815，respectively； the AUC of E_sd were 0.832， 0.857， 0.859， and 0.842，respectively；the sensitivity and specificity of E_max inside the lesion were 85.33% and 83.13%，respectively；the sensitivity and specificity of E_max at 1 mm around the lesion were 82.67% and 80.72%，respectively； the sensitivity and specificity at 2 mm around the lesion were 78.67% and 87.95%，respectively； the sensitivity and specificity at 3 mm around the lesion were 80.00% and 86.75%，respectively； the specificity of E_sd at 2 mm around the lesion was highest （93.33%）. Conclusion The SWE parameters inside and around the lesion and the manifestation of stiff rim sign can provide the reference for the differential diagnosis of benign and malignant breast NML， and have good diagnostic performance.

Objective To discuss the effectiveness of machine learning method combined with magnetic resonance diffusion weighted imaging （DWI） for recognition of the acute ischemic stroke （AIS） patient with onset time within 4.5 h，and to provide the reference for assisted assessment of onset time of AIS patients. Methods A total of 227 AIS patients with complete DWI imaging data were divided into onset time≤ 4.5 h group （n=70） and onset time >4.5 h group （n=157） based on their time from onset to DWI examination. The patients were randomly divided into training set （n=158） and test set （n=69） at a ratio of 7∶3. The regions of interest （ROI） were designated on the DWI images by ITK-SNAP annotation software， and the image features were extracted from the ROI images by the Pyradiomics package.The redundant features with high consistency were removed after evaluating the correlation of each feature by Spearman correlation test. Least absolute shrinkage and selection operator （LASSO） regression model with 10-fold cross-validation was used to recognize the image features of the AIS patients with onset time within 4.5 h. Seven machine learning algorithms， including Support Vector Machines （SVM）， K-Nearest Neighbors （KNN）， Decision Tree， Extra Trees， Random Forest， XGBoost， and LightGBM， were used to train the models. The performance of the models was evaluated by receiver operating characteristic curve （ROC） and the area under the curve （AUC）. Results A total of 107 image features， including 18 first-order features， 14 shape features， and 75 texture features， were extracted from the ROI images， among which 22 features were finally selected for recognition of the AIS patients with onset time within 4.5 h， including 4 first-order features， 6 shape features， and 12 texture features. The XGBoost model yielded the best results in recognizing the AIS patients with onset time within 4.5 h， and the AUC was 0.817， and the accuracy， sensitivity， and specificity were 0.739， 0.733， and 0.814，respectively. Conclusion The XGBoost model based on DWI imaging can effectively recognize the AIS patients with onset time within 4.5 h， which has certain clinical significance in assisting the assessment of onset time of the AIS patients.

Objective To discuss the method of primary culture and activity evaluation system of the neural stem cells （NSCs） of the fetal rats， and to confirm the optimal subculture conditions of the NSCs. Methods The SD rats with 11－14 d gestation were chosen，and the primary NSCs of the fetal rats were extracted. Nestin immunofluorescence staining was used to identify the NSCs. The NSCs were subcultured at the cell densities of 1.0×10⁴，2.0×10⁴，6.0×10⁴，1.0×10⁵， 1.6×10⁵ and 2.0×10⁵ mL^－1， then the morpholgy of the neurospheres was observed 48 h after passage and the diameter of the neurospheres was calculated. The survival rates of NSCs in the neurospheres with different diameters were detected by live-dead cell staining. Results The expression of nestin in the NSCs was positive， indicating that the cultured cells were the NSCs. The NSCs showed aggregation growth and strong cell-cell adhesion pattern forming neurospheres with an average diameter of （152.72±47.52） μm， and there were few single cells scattered between the neurospheres. Compared with 2.0×10⁴ mL^－1 subculture density，the diameters of the neurospheres at the subculture densities of differences 6.0×10⁴ mL^－1 and 1.0×10⁵ mL^－1 were increased （P<0.05）. There were no significant differences in the diameters of the neurospheres at the subculture densities of 1.0×10⁵ mL^－1， 1.6×10⁵ mL^－1， and 2.0×10⁵ mL^－1 （P>0.05）. Compared with neurospheres with diameters of 0—40 μm， 40—60 μm， 60—80 μm，and 80—100 μm， the survival rate of NSCs in the neurospheres with the diameters of 100—200 μm was decreased （P<0.05）. Conclusion The neurospheres with the diameters of 80—100 μm when subcultured at the density of 1.0×10⁵ mL^－1 can effectively improve the efficiency and activity of subculture of the NSCs.

Objective To improve the pre-processing laboratory test method for the severe acute respiratory syndome，coronavirus （SARS-CoV-2） specimens， and to evaluate its effectiveness. Methods A total of 90 SARS-CoV-2 negative specimens and 30 SARS-CoV-2 positive specimens were collected. The specimens were divided into traditional group， modified 1 group， and modified 2 group， according to the test method. The specimens in traditional group were inactivated by using the guanidine salts and a 56 ℃， 30 minute water bath， followed by single tube oscillation， specimen de-identification， and single-handed lid opening for testing. The specimens in modified 1 group were inactivated by using the guanidine salts， followed by single tube oscillation， specimen de-identification， and single-handed lid opening for testing. The specimens in modified 2 group were inactivated by using the guanidine salts， followed by batch oscillation in vertical， horizontal， and 180-degree angle， specimen de-identification， and single-handed lid opening for testing. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the cycle threshold （Ct） values and detection time of the specimens in various groups； Spearman correlation analysis was used to analyze the correlation of Ct values between various groups； Blank-Altman method was used to perform the consistency test； the detection time of SARS-CoV-2 of the specimens in various groups was recorded. Results The Ct values of the specimens in modified 1 group were strongly correlated with those in traditional group （P<0.01）， and the correlation coefficient（r）=0.975 4 （0.966 2－0.982 1）. The Ct values of the specimens in modified 2 group were strongly correlated with those in traditional group （P<0.01）， r=0.986 2 （0.980 9－0.990 0）. The inter-group deviation of detecting SARS-CoV-2 negative specimens between modified 1 group and traditional group was 0.158 2（－0.613 7－0.930 1）， and the inter-group deviation of detecting SARS-CoV-2 positive specimens was 0.117 0 （－0.403 1－0.637 1）， indicating good consistency.The inter-group deviation between modified 2 group and traditional group for detecting SARS-CoV-2 negative specimens was 0.015 7（－0.550 4－0.581 8）， and for detecting SARS-CoV-2 positive specimens was 0.022 7（－0.454 1－0.499 4）， which indicated the good consistency. The detection time of 90 SARS-CoV-2 specimens in modified 1 group was 15 min， while the detection time of 90 SARS-CoV-2 specimens in modified 2 group was 10 min and the detection time of 90 SARS-CoV-2 specimens in traditional group was 45 min. Compared with traditional group， the detection time in modified 1 group was reduced by 67%and the detection time in modified 2 group was reduced by 11% . Conclusion The modified SARS-CoV-2 specimen testing method shows strong correlation and good consistency with the traditional testing method. It can save the detection time and improve the efficiency.