Objective:To silence α-thalassemia/mental retardation syndrome X-linked gene(ATRX) in the cervical cancer HeLa cells, to detect the effect of ionizing radiation on the protein expressions of ATRX, γH2AX, Rad51 and γH2AX,Rad51 foci, and to explore the role of ATRX in DNA damage repair of the HeLa cells after irradiation. Methods:Three ATRX-shRNA and negative Control-shRNA lentiviral vectors were transfected into the 293T cells,and the lentiviruses were collected to infect the HeLa cells; puromycin was used to obtain the HeLa cells stably silencing ATRX named shA₁-HeLa, shA₂-HeLa, shA₃-HeLa, and shCon-HeLa; the silencing efficiency was detected by Western blotting method. After ionizing radiation, the expressions of ATRX, γH2AX, and Rad51 proteins were measured by Western blotting method, and the numbers of γH2AX and Rad51 foci in shCon-HeLa and shA₁-HeLa groups were observed and counted by immunofluorescence technique. Results:The ATRX protein expressed in shCon-HeLa cells, but did not express in shA₁-HeLa, shA₂-HeLa, and shA₃-HeLa cells; it indicated that the silencing efficiency was higher. At 1, 6, and 24 h after 2 and 8 Gy irradiation, the ATRX protein expression levels in shCon-HeLa group were increased gradually; it was most at 24 h, and the ATRX was highly expressed at 1, 6, and 24 h after 8 Gy irradiation. Compared with shCon-HeLa group,at 0-6 h after 4 Gy irradiation, the number of γH2AX foci in shA₁-HeLa group was significantly increased at 1 h (P<0.05), then was gradually decreased, but the number of γH2AX foci in shA₁-HeLa group was still higher at 6 h (P<0.01). The number of Rad51 foci was consistent with the changes of γH2AX focus number. Compared with shCon-HeLa group, the number of Rad51 foci was significantly increased at 1 h (P<0.05),and the number in shA₁-HeLa group was still higher at 6 h (P<0.01). At 0-16 h after 4 Gy irradiation, compared with shCon-HeLa, the expression amounts of γH2AX and Rad51 proteins in shA₁-HeLa group were increased. Conclusion:The HeLa cell models silencing ATRX are successfully obtained; ionizing radiation can cause the increase of ATRX expression level; the focus number and the protein expression amounts of γH2AX and Rad51 in HeLa cells silencing ATRX are higher than those in control group, which indicates that ATRX involves in the repair of radiation-induced DNA damage.

Objective:To study the effects of ursolic acid on the differentiation of cementoblast cell line OCCM-30 cells, and to provide the theoretical basis for the repair of root restoration. Methods:The OCCM-30 cells in logarithmic growth phase were divided into control group and different concentrations of ursolic acid groups. The OCCM-30 cells in ursolic acid groups were treated with different concentrations(0.625, 1.250 and 2.500 μmol·L^-1) of ursolic acid and the cells in control group did not receive any treatment. MTT method was performed to detect the inhibitory rates of proliferation of the OCCM-30 cells in various groups at different time points (24, 48, and 72 h); ALP (alkaline phosphatase) assay was performed to detect the cell differentiation and mineralization; real-time quantitative PCR was used to determine the expression levels of osteopontin(OPN) mRNA during 24 h; the expression levels of OPN protein at 3 and 5 d after administration were detected by Western blotting method. Results:There were no significant differences in the inhibitory rates of proliferation of OCCM-30 cells between control group and different concentrations of ursolic acid groups(P>0.05).Compared with control group, the ALP activities in the OCCM-30 cells in 2.500 μmol·L^-1 ursolic acid group at 3, 5, and 7 d after administration were significantly increased(P<0.05).Compared with control group,the expression levels of OPN mRNA and protein in the OCCM -30 cells in different concentrations of ursolic acid groups were significantly increased (P<0.01). Conclusion:Appropriate concentration of ursolic acid has no effect on the inhibitory rate of proliferation of OCCM-30 cells, but it can promote the differentiation of OCCM-30 cells and up-regulate the expression levels of OPN mRNA and protein.

Objective:To investigate the cardiovascular reflex induced by the activation of cardiac transient receptor potential vanillic acid receptor 1 (TRPV1), the central location of reflex and its peripheral neurotransmitters of the rats, and to elucidate the central and peripheral neural mechanisms of cardiovascular reflex mediated by TRPV1 receptor. Methods:A total of 60 healthy male SD rats were randomly divided into control group, ido-RTX group, atropine group, immunofluorescence group and nerve tract tracking group;there were 12 rats in each group. Pericardial intubation was performed in the rats in various groups after anesthesia. The changes of heart rates and blood pressures of the rats in control group,ido-RTX group and atropine group were observed by arterial intubation.The tissue samples were collected 1 h after intropericardial injection of capsaicin in imunofluorescence group,and the co-expression of c-Fos positive neurons induced by capsaicin and cholinesterase positive neurons in the medulla oblongata was observed by immunofluorescence technigue. Intraopericardial injected carbonyl cyanine fluorescent dye (Dil) was used as retrograde tracer in nerve tract group,and the distribution of Dil positive cells in the medulla oblongata was observed by fluorescence microscope 1 week later. Results:The blood pressure and heart rate of the rats in control group were decreased significantly after intrapericardial injection of capsaicin of the rats in control group (P<0.05).Compared with control group,the blood pressures and heart rates of the rats in ido-RTX group and atropine group had no significant changes after intrapericardial injection of capsaicin following intrapericardial injection of TRPV1 antagonist ido-RTX and intravenous injection of M receptor antagonist atropine(P>0.05); after intrapericardial injection of capsaicin, the number of c-Fos positive neurons in nucleus tractus solitarii,dorsal motor nucleus of vagus and nucleus ambiguous of medulla oblongata was increased significantly (P<0.05);most of these neurons were cholinesterase positive neurons; after intrapericardial injection of Dil, a large number of red fluorescent labeled neurons and nerve endings appeared in the nucleus tractus solitarii,dorsal motor nucleus of vagus and nucleus ambiguous. Conclusion:Intrapericardial injection of capsaicin can selectively activate the TRPV1 receptors in the heart, increase the excitability of cholinergic neurons in the cardiovascular center of the medulla oblongata, increase the peripheral release of acetylcholine, and produce an inhibitory effect on the cardiovascular activity by activating the M receptor.

Objective:To construct the miR-186 overexpression lentiviral vector and package the lentivirus, and toexplore the infection efficiency and the expression level of miR-186 in the HEK293T cells. Methods:The Hsa-miR-186 precursor sequence was used as a template to design and synthesize the primer, and the the pre-miR-186 gene was amplified by PCR. The pre-miR-186 gene sequence was cloned into the lentiviral vector FV040 carrying EGFP/Puromycin cassette. The recombinant lentiviral vector was digested by EcoRⅠand AgeⅠrestriction endonuclease and confirmed by sequencing. The recombinant FV040 Vector and FV040 miR-186 were co-transfected into the HEK293T cells with the helper plasmids using Lipofectamine 2000, respectively; the FV040 Vector lentivirus (control group) and the FV040 miR-186 lentivirus (experiment group) were collected and used to infect the HEK293T cells 48 h after transfection, respectively. The green fluorescence distribution in the HEK 293T cells was observed 48 h after transfection, and the expression level of miR-186 was determined by real-time fluorescence quantitative PCR. Results:The sequencing analysis results indicated that the sequence of miR-186 overexpressing lentivirus was identical with the sequence of miR-186 published on GenBank. The titers in control group and experiment group were 6×10⁸ TU·mL^-1 and 5×10⁸ TU·mL^-1, respectively. The relative expression level of miR-186 in the HEK293T cells in experiment group (12.640 0±0.788 4) was significantly increased by 15.07 times (t=14.72,P<0.01) compared with control group (0.838 7±0.145 6). Conclusion:The lentiviral vector which overexpresses miR-186 is constructed successfully and the miR-186 lentivirus is prepared. The HEK 293T cells are infected with miR-186 lentivirus suceessfully and the expression level of miR-186 in the HEK 293T cells is increased significantly.

Objective:To investigate the growth of Sporosarcina saromensis M52 obtained from the sediment samples in Xiamen,to locate the Cr(Ⅵ) reduetase, and to study the effects of metal ions and small molecules on the reducing ability of Cr(Ⅵ) resisistant strain M52. Methods:The seed solution of M52 strain was inoculated into the LB medium containing different concentrations (0-600 mg·L^-1) of Cr(Ⅵ). After cultivating for 0-48 h, the absorbance (A) value of M52 strain liquid at 600 nm was measured by UV spectrophotometry. The growth of M52 strain in LB medium containing 0, 50, 100, 200, 400, and 600 mg·L^-1 Cr(Ⅵ) was observed. The intracellular and extracellular active substances obtained by centrifugation of M52 bacteria solution before and after sonication and the M52 in control group were cultured at 37℃ and pH 7.5, respectively. Using diphenylcarbazide spectrophotometry, the concentrations of Cr(Ⅵ) in the solution at 0, 12, 24, and 48 h were measured, and the reduction rates of Cr(Ⅵ) in intracellular and extracellular active substances at each time point were calculated. 0.2 mmol·L^-1 Mn²⁺, Fe²⁺ and Cu²⁺, 1 mmol·L^-1 SDS and 1% Triton X-100, Tween 80 were added to the LB medium as treatment groups, and the untreated LB liquid medium was used as control group. The seed solution was inoculated in treatment groups and control group at 4% concentration. The reduction rates of Cr(Ⅵ) by M52 at 0, 6, 12, 24, 36, and 48 h were calculated. The changes of the reduction rates of Cr(Ⅵ) by M52 in metal ion and small molecule treatment groups were investigated. Results:When the concentration of Cr(Ⅵ) was lower than 100 mg·L^-1, the growth of strain was promoted with the increase of concentration; when the concentration of Cr(Ⅵ) was higher than 100 mg·L^-1, the growth of the strain was inhibited with the increase of concentration; when the concentration of Cr(Ⅵ) was higher than 600 mg·L^-1, the M52 strain hardly grew. Compared with control group, the reduction rates of Cr(Ⅵ) by M52 occurred both inside and outside the cells were increased(P<0.05),and the intracellular Cr(Ⅵ) had the highest reduction rate. Compared with control group, the reduction rates of Cr(Ⅵ) by M52 were increased in the presence of Cu²⁺ and Fe²⁺ (P<0.05), and Cu²⁺ >Fe²⁺; the reduction rate was reduced in the presence of Mn²⁺ (P<0.05). Compared with control group, the reduction rates of Cr(Ⅵ) by M52 were reduced in the presence of small molecules SDS, Triton X-100, and Tween 80, and SDS > Triton X-100 > Tween 80. Conclusion:Low concentration of Cr(Ⅵ) can promote the growth of the M52 strain,and high concentration of Cr(Ⅵ) can inhibit the growth of the strain. The reduction of Cr(Ⅵ) by M52 mainly occurs in the cells. Cu²⁺ and Fe²⁺ can promote the reduction of Cr(Ⅵ) by M52.Tween 80, Triton X-100, and SDS can inhibit the reduction of Cr(Ⅵ) by M52.

Objective:To investigate the inhibitory effect of dehydroandrographolide (DA) on the hepatocyte apoptosis induced by carbon tetrachloride (CCl₄) in the hepatic fibrosis model mice, and to elucidate its possible mechanism. Methods:A total of 30 male C57BL/6J mice aged 6-8 weeks were randomly divided into control group, model group and treatment group;there were 10 mice in each group. Except the normal control group, the mice in other two groups were intraperitoneally injected with 20% CCl₄ 2.0 mL·kg^-1, three times a week;the hepatic fibrosis models were establised. The mice in control group were given the same volume of olive oil. The mice in treatment group were intragastrically given 100 mg·kg^-1 DA,three times a week; while the mice in control group and model group were given the equal volume of normal saline. Four weeks later, 24 h after the last administration, the eyeball blood was taken, the marrow was broken, and the liver was taken. The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of the mice in various groups were detected; the activities of superoxide dismutase (SOD) and the levels of malondialdehyde (MDA) in liver tissue of the mice in various groups were detected. The pathological performance of liver tissue of the mice in various groups were observed by HE staining and Sirius red staining. Western blotting method was used to detect the levels of 8-oxoguanine DNA glycosylase 1 (Ogg1), Caspase-3, Bcl-2-associated X protein(Bax), and B-cell lymphoma-2 (Bcl-2) proteins in liver tissue of the mice in various groups. The expression levels of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) proteins in liver tissue of the mice in various groups were observed by immunohistochemistry. Results:Compared with control group, the activities of serum ALT and AST and the level of MDA in liver tissue of the mice in model group were significantly increased (P<0.05), and the activity of SOD in liver tissue was significantly decreased (P<0.05). Compared with model group, the activities of ALT and AST in serum and the level of MDA in liver tissue of the mice in treatment group were significantly decreased (P<0.05), and the activity of SOD in liver tissue was significantly increased (P<0.05). The HE staining and Sirius red staining results showed that compared with model group, the inflammatory cell infiltration and collagen deposition in liver tissue of the mice in treatment group were significantly improved. Compared with control group, the expression levels of Ogg1, Caspase-3, Bax, TGF-β1,α-SMA and Bcl-2 proteins in liver tissue of the mice in treatment group were significantly increased (P<0.05); compared with model group, the expression levels of Ogg1, Caspase-3, Bax, TGF-β1 and α-SMA proteins in liver tissue of the mice in treatment group were significantly decreased (P<0.05), and the expression level of Bcl-2 protein was significantly increased (P<0.05). Conclusion:DA has a protective effect in the mice with hepatic fibrosis induced by CCl₄, which might be related to reducing oxidative stress injury, inhibiting hepatocyte apoptosis and reducing hepatic stellate cell activation.

Objective:To construct a cDNA library of human promyelocytic leukemia (HL60), and to elucidate the mechanism of related genes in the pathogenesis and development of acute promyelocytic leukemia (APL). Methods:The total RNA of HL60 cells was extracted by Trizol method.The mRNA of samples were isolated and purified by centrifugal column method. The first strand of the cDNA was synthesized by reverse transcriptase-polymerase chain reaction (RT-PCR) while the second strand was synthesized by enzyme-catalyzed method. The cDNA fragments were recovered and then linked to the pPR3-N vector. The human promyelocytic cDNA library was constructed by homologous recombination in yeast strain Y187. The culture fluid was coated with LB plate to identify the library capacity, and PCR method was used to identify the size and distribution of the inserted fragment. Results:The RNA strand extracted from HL60 cells was clear, non-degradable and had low dispersion. The purified cell mRNA was used as template to synthesize the cDNA. After recovering the cDNA fragments, the cDNA fragments were successfully linked to the pPR3-N vector. The recombinant vector was transformed into yeast strain Y187, and the human promyelocytic cDNA library was successfully constructed by homologous recombination. The original electro transformation bacteria were diluted 100 times and 10 μL coating plate was taken, about 250 clones were generated. The total library capacity was 2.5×10⁶ CFU mL^-1 and the total library capacity was 1.25×10⁷CFU for 5 mL of transformed original bacterial solution. The average insertion fragment was more than 1 200 bp, and the positive rate was 100%. Conclusion:The human promyelocyic cDNA library is successfully constructed, and it lays a foundation for studying the pathogenesis and therapeutic mechanism of leukemia.

Objective:To investigate the effects of extractum trametes robiniophila murr on the filtration rate, motility and cytoskeleton rearrangement of the podocytes in vitro of the puromycin aminonucleoside(PAN)-treated mice, and to clarify the protective effect of extractum trametes robiniophila murr on the podocyte injury and its mechanism. Methods:The podocytes cultured in vitro were randomly divided into control group, model group and test group. The podocytess in model group were treated with 50 mg·L^-1 PAN for 24 h; the podocytess in test group were treated with 10 g·L^-1 extractum trametes robiniophila murr for 1 h and then treated with 50 mg·L^-1 PAN for 24 h. The filtration rate of podocytes to FITC-BSA was measured by two-compartment diffusion system; the scratch repair rate of podocytes was detected by cell scratch test, and the number of podocytes passing through the membrane was measured by Transwell cell migration test. Laser confocal microscope was used to observe the cytoskeleton rearrangement of podocyte cytoskeleton protein F-actin labeled with Invitrogen phalloidin directly. Results:Compared with control group, the FITC-BSA filtration rate of the podocytes in model group was increased significantly(P<0.01);compared with model group, the FITC-BSA filtration rate of the podocytes in test group was decreased significantly(P<0.01).Compared with control group, the scratch repair rate of podocytes and the number of transmembrane cells in model group were transmembrane(P<0.05);compared with model group, the scratch repair rate of podocytes and the number of migration migration cells in test group were decreased significantly(P<0.05). Compared with control group, the expression level of F-actin in the podocytes in model group was decreased significantly(P<0.01), the rearrangement rate of F-actin was increased signifiantly(P<0.01),and the structure of podocyte cytoskeleton was disordered;compared with model group, the expression level of F-actin in the podocytes in test group was increased significantly(P<0.01), the rearrangement rate of F-actin was decreased significantly(P<0.01),and the skeleton rearrangement was alleviated obviously. Conclusion:Extractum trametes robiniophila murr could reduce the filtration rate of podocytes to BSA in vitro under the pathological condition,and its possible mechanism is that extractum trametes robiniophila murr reduces the motility of podocytes and improve the rearrangement of podocyte cytoskeleton in vitro.

Objective:To observe the orthodontic tooth movement after regeneration of Beagl dog's periodontal tissue defect with biphasic calcium phosphate(BCP), and to elucidate the mechanism of BCP as a scaffold material for periodontal regeneration. Methods:Six adult male Beagle dogs were selected;the right upper quardrant(B) and the left lower quardrant(D) regions of each dog were used as blank control group, and the left upper quardrant(A) and the right lower quardrant(D) regions were used to establish the dog models of periodontal tissue defect of bilateral incisors. BCP was implanted into the defect to regenerate the defect. After 12 weeks of BCP implantation into the defect for defect tissue regeneration, the orthodontic tooth movement models were established (experimental group). Stress stimulation was applied in experimental group and control group, respectively. Two Beagle dogs were randomly executed at 1, 2 and 4 weeks after loading. The specimens were stained, the histological changes of regenerated periodontal tissue and the expression of core binding factor α1(Cbfα1) regulating osteogenesis under stress were observed;the changes in normal periodontal tissue under the same stress were observed. Results:The Masson staining results showed that the periodontal ligament of the dogs in experimental group and control group became narrower and denser at 1 week after stress stimulation; the alveolar bone of the dogs in dogs in experimental group was mainly blue; the alveolar bone of the dogs in control group was generally red except for the compression area of the periodontal ligament. At 4 weeks after stress stimulation, the alveolar bone of the dogs in experimental group was red-blue, while the alveolar bone of the dogs in control group was red; the blood vessels in periodontal ligament of the dogs in two groups were rounded. One week after stress stimulation, the alveolar bone surface of the dogs in experimental group and control group was covered with bone resorption lacunae containing the multinucleated osteoclasts, the cytoplasmic Cbfα1 staining was positive, and the compressed and deformed periodontal ligament cells were arranged disorderly. Four weeks after stress stimulation, the osteoclasts disappeared in experimental group, and the bone resorption lacunae was filled by osteoblasts, and the osteoblasts on the other side of the trabecula were also active; the expression of Cbfα1 was still found in the bone marrow mesenchymal cells and the marginal osteoblasts of alveolar bone. The expression of Cbfα1 was weak in control group. Conclusion:The periodontal tissue regenerated by BCP has reached the normal periodontal tissue. Under the action of orthodontic force, it has normal osteogenesis function and can complete the bone remodeling process of orthodontic tooth movement.

Objective:To investigate the inhibitory effect of coenzyme Q10 (Co-Q10) on the apoptosis of human coronary endothelial cells (HCAECs) induced by highglucose, and to elucidate its possible mechanism. Methods:The HCAECs were divided into control group, high glucose group and high glucose combined with 5, 10,and20 μmol·L^-1 Co-Q10 treatment groups;the HCAECs in control group were cultured for 24 h using a routine culture method. The cells in high glucose group were treated with 30 mmol·L^-1 glucose for 24 h;the cells in high glucose combined with 5, 10,and 20 μmol·L^-1 Co-Q10 treatment groups were treated with 5, 10,and 20 μmol·L^-1 Co-Q10 combined with 30 mmol·L^-1 glucose for 24 h, respectively. The cell viabilities of HCAECs in various groups were measured by CCK-8 assay. The apoptotic rates of HCAECs in high glucose group and high glucose combined with 10 μmol·L^-1 Co-Q10 treatment group were detected by Hoechst-PI double staining. The cell mitochondrial membrane potentials of HCAECs in high glucose group and high glucose combined with 10 μmol·L^-1 Co-Q10 treatment group were determined by Mito-tracker staining.The mitochondrial reative oxygen species (mtROS) levels in the HCAECs in high glucose group and high glucose combined with 10 μmol·L^-1 Co-Q10 treatment group were measured by MitoSox staining. The protein expression levels of B cell lymphoma/leukemia 2 protein (Bcl-2), Bcl-2 assaciated X protein (Bax), Bcl-2 assaciated death promoter (Bad) and X-linked inhibitor of apoptosis protein (x-IAP) in the HCAECs in high glucose group and high glucose combined with 10 μmol·L^-1 Co-Q10 treatment group were detected by Western blotting method. Results:Compared with control group, the cell viability of HCAECs in high glucose group was significantly reduced (P<0.01); compared with high glucose group, the cell viabilities of HCAECs in high glucose combined with 5, 10,and 20 μmol·L^-1 Co-Q10 treatment groups were significantly increased(P<0.05 or P<0.01), especially in high glucose combined with 10 μmol·L^-1 Co-Q10 treatment group (P<0.01). Compared with high glucose group, the apoptotic rate, the mitochondrial membrane potential and the mtROS level of HCAECs in high glucose combined with 10 μmol·L^-1 Co-Q10 treatment group were significantly decreased (P<0.01).The Western blotting results showed that compared with high glucose group, the expression levels of Bax and Bad proteins in the HCAECs in high glucose combined with 10 μmol·L^-1 Co-Q10 treatment group were decreased significantly(P<0.01), and the expression levels of Bcl-2 and x-IAP proteins were increased significantly (P<0.01). Conclusion:Co-Q10 may reduce the apoptosis of HCAECs induced by high glucose through inhibiting the mitochondrial apoptosis-related pathway to ptotect the cells.

Objective:To investigate the effects of silencing zinc finger E-box binding homeobox1(ZEB1) gene on the expressions of mesenchymal markers and cell migration in the glioma U87 cells, and to clarify the effect of ZEB1 on the epithelial to mesenchymal transition (EMT) in the glioma cells. Methods:The constructed ZEB1 shRNA interfering plasmid and control plasmid (shCtrl) were transfected into the glioma U87 cells and the interfering effects were detected by Western blotting method. The glioma U87 cells were divided into control group (the glioma U87 cells were transfected with shCtrl), EMT group (EMT was induced by TGF-β1 in the glioma U87 cells transfected with shCtrl) and ZEB1 silence group (EMT was induced by TGF-β1 in the glioma U87 cells transfected with ZEB1 shRNAs plasmid). The protein expression levels of mesenchymal markers (N-cadherin, Vimentin), and matrix metalloproteinase-9 (MMP-9) in the glioma U87 cells were measured by Western blotting method. The scratch-healing assay was performed to examine the migration ability of glioma cells. Results:The Western blotting results showed that the expression levels of ZEB1 in the glioma U87 cells transfected with shZEB1#1 and shZEB1#2 were significantly lower than that in the cells transfected with shCtrl (P<0.05 or P<0.01), and the inhibitory effect of shZEB1#2 on the ZEB1 expression was more obvious, indicating that ZEB1 was stably transfected into the U87 cells. Compared with control group, the expression levels of mesenchymal markers N-cadherin, Vimentin, and MMP-9 in EMT group were significantly increased (P<0.05 or P<0.01).Compared with EMT group, the expression levels of the above proteins in ZEB1 silencing group were markedly reduced (P<0.05 or P<0.01).The cell migration rate in EMT group was obviously elevated compared with control group (P<0.01), and the cell migration rate of the glioma U87 cells in ZEB1 silence group was significantly lower than that in EMT group(P<0.01). Conclusion:Silencing ZEB1 gene expression can inhibit the EMT in the glioma U87 cells and reduce the cell migration abilities, suggesting ZEB1 as an important therapeutic target of invasive glioma.

Objective:To observe the effect of 17β-estradiol (17β-E2) on the calcium (Ca²⁺) channels during the osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs), and to elucidate the mechanism of 17β-E2 in the osteogenic differentiation of MSCs. Methods:The MSCs were separated by density gradient centrifugation and adherent screening, and passaged for 3 times continuously to induce osteoblast differentiation. The MSCs were divided into control group[cultivated in osteoblast culture medium alone (OBM)] and different doses of 17β-E2 groups(added with 0.1, 1.0, 10.0, and 100.0 pmol·L^-1 17β-E2 in OBM, respectively). On the 14th day of osteogenic induction, the cells in each group were stained with Fluo-3/AM, and the Ca²⁺ levels were determinated by laser scanning confocal microscope;the mean fluorescence intensity (MFI) was used to respresent the level of Ca²⁺. Whole-cell Ca²⁺ currents were recorded using whole-cell patch clamp technique under different conditions. Results:The MSCs with fibroblast-like cells, oval nuclei and visible nucleoli were successfully isolated by density gradient centrifugation and adherent screening. The subcultured MSCs grew vigorously and maintained the morphological characteristics of primary cells. Following the increase of 17β-E2 concentration,the Fluo-3 fluorescence staining intensity of Ca²⁺ in each group was also gradually increased, especially in 100.0 pmol·L^-1 17β-E2 group. Compared with control group, the MFI of Ca²⁺ and the current peak values of Ca²⁺ in 10.0 and 100.0 pmol·L^-1 17β-E2 groups were increased (P<0.05 or P<0.01) during osteogenic differentiation of the MSCs; the MFI of Ca²⁺ and the current peak values of Ca²⁺ in 0.1 and 1.0 pmol·L^-1 17β-E2 groups showed no significant differences (P>0.05). Conclusion:The cells isolated by density gradient and adherent screening method are the rat MScs. 17β-E2 plays a role in promoting osteogenesis by enhancing the opening of Ca²⁺ channels in the MSCs and the inward current of calcium ions in a dose-dependent manner.

Objective:To discuss the role for down-regulation of JNK enzymatic activity in the tumor treatments by interrupting JNK gene expression with JNKinhibitor SP600125 and JNK-siRNA encapsulated by lipid nanoparticles in vivo and in vitro. Methods:In vitro experiments, the experiment was divided into siRNA group and inhibitor SP600125 group. In siRNA group,JNK-siRNA and NC-siRNA were transfected into the human prostate cancer cells (PC cells), human hepatocellular carcinoma cells(SMMC-7721 cells) and human breast cancer cells(MCF cells), respectively. In inhibitor SP600125 group, SP600125 was delivered to human hepatocellular carcinoma cells. The expression levels of JNK or p-JNK proteins in human prostate cancer cells,human hepatocellular carcinoma cells,and human breast cancer cells after transfected with JNK-siRNA and inhibitor SP600125 were detected by Western blotting method. The cell viabilities of tumor cells in various groups were examined by WST-1 proliferation assay. In vivo experiments,the human hepatocellular carcinoma SMMC-7721 cells were used to establish the subcutaneous hepatocellular carcinoma xenograft tumor models by subcutaneous injection. The eight mice were fed until the tumors were generated to 3 mm×3 mm. They were randomly divided into inhibitor SP600125 group and negative control group,JNK-siRNA group and NC-siRNA control group. Each mouse in various groups was injected intratumorally with 5 nmol SP600125,negative control solution, lipid nanoparticles-mediated JNK-siRNA and NC-siRNA negative control. The volumes of tumors of the mice in various groups were observed. The expressions of JNK or p-JNK proteins in tumor tissue were examined by immunohistochemical staining. Results: In vitro experiments, compared with NC-siRNA control group, the expression level of JNK protein in human prostate cancer cells, human hepatocellular carcinoma cells and human breast cancer cells in JNK-siRNA group were decreased(P<0.01); the expression level of p-JNK protein in human hepatocellular carcinoma cells in inhibitor SP600125 group was significantly increased compared with its negative control group(P<0.01).In vivo experiment, the human hepatocellular carcinoma cells were taken as an example, the volume of tumor of the mice in inhibitor SP600125 group was significantly reduced compared with negative control group, whereas the change of tumor volume in JNK-siRNAs group was not significant compared with NC-siRNA control group. The immunohistochemical staining results showed that compared with their negative control groups, the expression amount of p-JNK protein in tumor tissue of the mice in SP600125 group and the expression amount of JNK protein in tumor tissue of the mice in JNK-siRNA group were decreased (P<0.01). Conclusion:Down-regulation of enzymatic activity of JNK can decrease the expression level of JNK gene and then inhibit the tumor growth both in vivo and in vitro.

Objective:To investigate the effect of high salt diet on the arterial blood pressure in the urea transporter B(UT-B) gene depletion (UT-B ^-/-) mice, and to clarify the possible mechanism of the UT-B^-/- leading to the changes in the arterial blood pressure of the mice. Methods:The heterozygous (UT-B^+/-) mice were mated to obtain the wild-type (UT-B^+/+) and UT-B^-/- mice with the same genetic background. The 4-week-old male UT-B^+/+ and UT-B^-/- mice were selected and fed on normal diet (0.3% NaCl) or high salt diet (8.0% NaCl) for 4 weeks. The mice were divided into UT-B^+/+ mice+normal diet (UT-B^+/++N) group,UT-B^-/- mice +normal diet (UT-B^-/-+N) group,UT-B^+/+ mice+high salt diet(UT-B^+/++H) group,and UT-B^-/- mice+high salt diet(UT-B^-/-+H) group. The changes in water intakes and mean arterial pressures of the mice in various groups were monitored; RT-PCR, Western blotting and immunohistochemistry were used to detect the expression levels and location of UT-B mRNA and protein in choroid plexus(CP) of the brain tissue of the mice. The levels of serum angiotensin Ⅱ (Ang Ⅱ) and the Na⁺ levels in cerebrospinal fluid of the mice in various groups were determined by ELISA. Results:The PCR results of genomic DNA of mouse tail showed that there was a 400 bp base fragment in the UT-B^+/+ mice, 250 and 400 bp base fragments in the UT-B^+/- mice, and 250 bp base fragment in the UT-B^-/- mice. Compared with normal salt diet group, the water intake of the mice in high salt diet was significantly increased (P<0.01); compared with UT-B^-/-+N group and UT-B^+/++H group,the mean arterial pressure of the mice in UT-B^-/-+H group was significantly increased(P<0.01).The UT-B mRNA and protein expressed in the epithelial cells of CP in the UT-B^+/+ mice. Compared with UT-B^-/-+N group and UT-B^+/+ mice+H group, the Ang Ⅱ level in serum of the mice in UT-B^-/- mice+H group was significantly increased (P<0.01); the Na⁺ level in cerebrospinal fluid of the mice was significantly increased (P<0.05). Conclusion:High salt diet can cause a significant increase in the mean arterial pressure in the UT-B^-/- mice, and its mechanism is related to increasing the serum Ang Ⅱ level and the Na⁺ level in cerebrospinal fluid in the mice.

Objective:To establish the rat models of myocardial ischemia reperfusion-related no-reflow under non-artificial ventilator, to evaluate the models by morphology, hematological biochemistry and hemorheology, and to lay the foundation for studying the pathogenesis of cardiovascular diseases and evaluating the pharmacodynamics of drugs of cardiovascular diseases. Methods:Fifty healthy female Wistar rats were randomly divided into sham operation group (n=20) and model group (n=30). The myocardial ischemia reperfusion-related no-reflow rat models in model group were induced by ligation of left anterior descending coronary artery for 2 h and another 2 h for reperfusion. The rats in sham group were only threaded and not ligated. The model was evaluated by detecting the area at risk (AAR), area at infarct (AAI), area at no-reflow (AAN) of myocardium, the activities of creatine kinase MB (CK-MB), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) in serum,the whole blood viscosity,the plasma viscosity, the platelet adhesion rate (PAR),the platelet aggregation (PAG) of the rats. Results:Compared with sham operation group, the AAR, AAI,and AAN of myocardium, the activities of CK-MB, LDH,and AST in serum,the whole blood low shear viscosity(20/s),the middle shear viscosity(60/s), the high shear viscosity(120/s),the plasma viscosity, PAR, PAG (1, 3, 5 min)and the maximum platelet aggregation rate(MAPG) of the rats in model group were significantly increased (P<0.05 or P<0.01). Conclusion:The rat model of myocardial ischemia reperfusion-related no-reflow under non-artificial ventilator is successfully established. This method is characterized by simple operation, decreased injury to animals, higher stability of model construction, shorter experimental cycle and less cost.

Objective:To explore the effect of Helicobacter pylori virulence factor cytotoxin-associated gene A protein (CagA) on the extracellular regulated protein kinase (ERK) signaling pathway, and to elucidate the carcinogenesis mechanism of CagA. Methods:The pcDNA3.1/CagA eukaryotic expression vector was constructed, and the gastric cancer AGS cells were divided into blank control group (blank vector transfection), CagA transfection group (GZ7/CagA transfection), and CagA + ERKi group (ERK1/2 inhibitor pretreatment + GZ7/CagA transfection).The expression levels of CagA, phosphorylated ERK (p-ERK), total ERK (T-ERK), B-lymphocytoma-2 (Bcl-2), Bcl-2-related X protein (Bax) and cleaved caspase-3 proteins in the cells in various groups were determined by Western blotting method. The activities of AGS cells in various groups were determined by CCK-8 method, and the apoptotic rates of AGS cells were determined by flow cytometry. Results:Compared with blank control group, the expression levels of CagA, p-ERK, and Bcl-2 proteins in CagA transfection group were significantly increased(P<0.01), and the expression levels of Bax and cleaved caspase-3 proteins were significantly decreased (P<0.01). Compared with CagA transfection group, the expression levels of p-ERK and Bcl-2 proteins in CagA+ERKi group were significantly decreased (P<0.01), and the expression levels of Bax and cleaved caspase-3 proteins were significantly increased (P<0.01). Compared with CagA transfection group, the activities of gastric cancer cells in CagA + ERKi group at different time points were significantly decreased (P<0.01). Compared with CagA transfection group, the apoptotic rate of gastric cancer cells in CagA + ERKi group was significantly increased (P<0.05). Conclusion:Helicobacter pylori virulence factor CagA can inhibit the proliferation of gastric cancer cells and promote the apoptosis of gastric cancer cells, and its mechanism may be related to the activation of ERK signaling pathway by CagA.

Objective:To investigate the therapeutic effect of ceftriaxone in the rats with subarachnoid hemorrhage(SAH), and to clarify its mechanism. Methods:A total of 48 adult male SD rats were randomly divided into sham operation group, SAH group, 3-methyl adenine (3-MA) group and ceftriaxone (CEF) group; there were 12 rats in each group. The SAH models were established by intravascular puncture;the rats were administered intraperitoneally;the dose of 3-MA was 15 mg·kg^-1, and the dose of CEF was 500 mg·kg^-1. The rats were sacrificed 24 h after model establishment, and the pathology of brain tissue of the rats was observed by HE staining. The apoptosis of neurons was detected by TUNEL staining. The number of autophagy-related proteins Beclin-1 and LC3-Ⅱ and the apoptosis factor Caspase-3 protein in hippocampus tissue of the rats in various groups were detected by immunohistochemistry and Western blotting methods. Results:Compared with SAH group, the neurological score of the rats in CEF group was significantly increased (P<0.01). The HE staining results showed the nerve cells in the hippocampus area of the rats in SAH group were swollen with loose cytoplasm and had obvious nuclear condensation,fragmentation and dissolution;compared with SAH group,the neural degeneration of the rats in CEF group was improved, and more normal nerve tissue morphology appeared; while the nerve cell damage in 3-MA group was more serious, the dead nerve cells were in full filed, and the cell layers were disordered. The quantitative analysis results showed that the number of necrotic nerve cells in CEF group was significantly lower than that in SAH,and the number of necrotic nerve cells in 3-MA group was significantly higher than that in SAH group (P<0.05). The immunohistochemical staining results showed that the number of Beclin-1 and LC3-Ⅱ posivive cells in hippocampus tissue of the rats in CEF group was significantly higher than that in SAH group (P<0.05); the number of Beclin-1 and LC3-Ⅱ positive cells in 3-MA group was significantly lower than that in SAH group (P<0.05). The TUNEL staining resluts showed that the number of apoptotic nerve cells in CEF group was significantly lower than that in SAH group (P<0.05),and the number of apoptotic nerve cells in 3-MA group was higher than that in SAH group(P<0.05). The Western blotting results showed that the expression level of Caspase-3 protein in hippocampus tissue of the rats in CEF group was significantly lower than that in SAH group (P<0.05), while the expression level of Caspase-3 protein in 3-MA group was significantly higher than that in SAH group (P<0.05). Conclusion:CEF has the protective effect on the nerve injury of the rats with SAH, and its mechanism may be related to up-regulation of autophagy and reduction of apoptosis of the neurons.

Objective:To establish models of young periapical periodontitis in the Beagle dogs and to explore the effects of nerve growth factor (NGF) on the regeneration of young permanent teeth pulp tissue of the Beagle dogs, and clarify the action mechanism. Methods:Twelve permanent teeth of 6 Beagle dogs were selected and divided into control group, hydrogel group, and hydrogel combined with NGF group, with 4 permanent dental of 2 Beagle dogs in each group.The teeth in control group were not treated.The teeth in hydrogel group were established the periapical periodontitis models and were treated with hydrogel alone.The teeth in hydrogel combined with NGF group were established the models of periapical periodontitis and treated with NGF combined with hydrogel.The histological changes of the experimental teeth in various groups were observed by HE staining.The depth of regenerated tissue and the area of regenerated tissue of the experimental teeth in various groups were measured. Results:The young permanent periapical periodontitis models of Beagle dogs were successfully established.In control group,the dental pulp tissue in the root canal was normal, and it was composed of fibroblasts, odontoblasts and blood vessels;in hydrogel group,there was no new hard tissue formed at the apex, and fiber repair occured at the apical period;in hydrogel combined with NGF group, there was newly formed hard tissue in the root canal,the cementoblast-like cells and the cementum matrix lacuna could be seen,the free cementum-like tissue was seen in the root canal,and the newly formed soft tissue in the root canal was mainly the blood vessels and the fibroblasts. The depth of regeneranted tissue and the area of regenerated tissue in hydrogel combined with NGF group were higher than those in hydrogel group (t=66.261,P<0.01;t=60.363,P<0.01). Conclusion:NGF has the effect of promoting the regeneration of dental pulp tissue of young permanent periapical periodontitis of the Beagle dogs.

Objective:To explore the effects of baicalin on the cognitive function and the expression of vascular endothelial growth factor (VEGF), and endostatin(ES) in the brain tissue of the cerebral small vessel disease model rats, and to clarify their mechanisms. Methods:The rat models of cerebellar small vessel disease were duplicated by in vitro implantation of allogeneic microemboli. After successfully modeling, 48 model rats were randomly divided into model group, low dose(50 mg·kg^-1) of baicalin group, and high dose (100 mg·kg^-1) of baicalin group(n=16);On and the sham operation rats were used as control group(n=16).On the first day after modeling, the rats in each group were given drugs,and the learning and memory function of the rats in various groups were detected by Morris water maze experiment at 7, 14, and 28 d after modeling, respectively. The expression levels of VEGF and ES mRNA and protein in the brain tissue of the rats in various groups were detected by RT-PCR and Western blotting methods, and the morphology of the hippocampus tissue of the brain of the rats in various groups were observed by HE staining; the expressions of VEGF and ES proteins in the hippocampus tissue of the brain of the rats in various groups were detected by immunohistochemistry. Results:Compared with control group, the escape latency in Morris water maze of the rats in model group was significantly prolonged(P<0.01), the number of crossing the platform was significantly reduced (P<0.01), the brain tissue lesion was severe, the expression levels of VEGF mRNA and protein in brain tissue were significantly decreased (P<0.01), and the expression levels of ES mRNA and protein were significantly increased (P<0.01). Compared with model group, the escape latency in Morris water maze of the rats in high dose of baicalin group was significantly shortened(P<0.01), the number of crossing the platform was significantly increased(P<0.01), the degree of brain tissue lesion was alleviated, the expression levels of VEGF mRNA and protein in brain tissue were significantly increased(P<0.01), and the expression levels of ES mRNA and protein were significantly decreased (P<0.01). Conclusion:Baicalin can reduce the escape latency, improve the pathological changes, and repaire the cognitive function of brain tissue; it can decrease the VEGF expression level and increase the expression lvels of ES in the brain tissue of the cerebral small vessel disease model rats, and plays a protective role in the brain tissue.

Objective:To investigate the effects of butylphthalide on the hippocampal neuron apoptosis and p38 mitogen-activated protein kinase (MAPK) signaling pathway in the rats with ischemic stroke, and to elucidate the mechanism of butylphthalide in ischemic stroke. Methods:A total of 102 male rats were randomly divided into sham operation group, model group and butylphthalide group;there were 34 rats in each group. The rats in model group and butylphthalide group were used to establish the focal cerebral ischemia models with modified Zea-Longa method. The rats in butylphthalide group were treated with butylphthalide (4.5 mg·kg^-1) after modeling.The rats in sham operation group were intraperitoneally injected with the same volume of normal saline at the same time points. The morphology of neurons was observed by HE staining. The apoptosis of hippocampal neurons was observed by in situ terminal transferase labeling (TUNEL) staining. The expression levels of activated caspase-3 (cleaved caspase-3), B lymphocyte tumor-2 (Bcl-2), Bcl-2 related X protein (Bax), p38, phosphorylation-p38 (p-P38) and MAPK proteins in hippocampus tissue of the rats in various groups were determined by Western blotting method. The expression levels of cleaved caspase-3, Bax, Bcl-2, p38, and MAPK mRNA in hippocampus tissue of the rats in various groups were determined by reverse transcription-polymerase chain reaction (RT-PCR). Results:The neurons in the hippocampal CA1 area of the rats in sham operation group were well aligned and the nuclei and cell membrane were normal. In model group, the neurons in the hippocampal CA1 area were disordered, the cells were swollen and burst, and the nuclei had pyknosis. In butylphthalide group, the structure of some neurons in the hippocampal CA1 area returned to normal. Compared with sham operation group, the number of neurons in the hippocampal CA1 area of the rats in model group was significantly reduced (P<0.01), and the apoptotic index of neurons was significantly increased (P<0.01).Compared with model group, the number of neurons in hippocampal CA1 area of the rats in butylphthalide group was significantly increased (P<0.01), and the apoptotic index of neurons was significantly decreased (P<0.01). Compared with sham operation group, the expression levels of cleaved caspase-3, Bax, p-p38 and MAPK proteins in hippocampal tissue of the rats in model group were significantly increased (P<0.01), and the expression level of Bcl-2 protein in hippocampal tissue of the rats in model group was significantly decreased (P<0.01); compared with model group, the expression levels of cleaved caspase-3, Bax, p-p38 and MAPK proteins in hippocampus tissue of the rats in butylphthalide group were significantly decreased (P<0.01),and the expression level of Bcl-2 protein in hippocampus tissue of the rats in butylphthalide group was significantly increased (P<0.01). Compared with sham operation group, the expression levels of cleaved caspase-3, Bax and MAPK mRNA in hippocampus tissue of the rats in model group were significantly increased (P<0.01), and the expression level of Bcl-2 mRNA in hippocampal tissue of the rats in model group was significantly decreased (P<0.01); compared with model group, the expression levels of cleaved caspase-3, Bax and MAPK mRNA in hippocampus tissue of the rats in butylphthalide group were significantly decreased (P<0.01),and the expression level of Bcl-2 mRNA in hippocampus tissue of the rats in butylphthalide group was significantly increased (P<0.01). Conclusion:Butylphthalide can inhibit the apoptosis of hippocampal neurons in the rats with ischemic stroke by inhibiting the p38 MAPK signaling pathway in the hippocampus tissue.

Objective::To investigate the relationship between the expression level of fibrinogen (Fib) and the prognosis of breast cancer patients with Meta-analysis, and to provide evidence-based medical evidence for the judgement of prognosis of the breast cancer patients. Methods:PubMed, EMbase, Cochrane Library and CNKI databases were searched for collecting the studies about the relationship between Fib and prognosis of the breast cancer patients from inception to January 2019. Two reviewers extracted the data according to inclusion and exclusion criteria. Meta-analysis was performed by using Stata 14.0 software. Results:A total of 158 204 patients from 8 case-control studies were included.The heterogeneity test results included in the studies were I²=58.4%,P=0.019, and the data were analyzed using a random effect model. The results suggested that high expression level of Fib was associated with the lower overall survival (OS) in the breast cancer patients(HR=1.37, 95%CI:1.14,1.64, Z=3.36,P=0.001).The high expression level of Fib was associated with the lower OS in the Asia breast cancer patients(HR=1.83, 95%CI:1.23,2.72, Z=3.00,P=0.003).The sensitivity analysis results showed that the results of 8 included studies were stable and reliable; Begg's test, Z=1.61,P=0.108, suggesting that there was no obvious publication bias. Conclusion:High expression level of Fib is significantly associated with the poor prognosis in the breast cancer patients.

Objective:To analyze the differentially expressed miRNAs in triple negative breast cancer (TNBC) and predict their target genes through The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, to explore their biological functions and molecular mechanisms, and to find the prognosis-related targets of TNBC. Methods:A total of 343 miRNAs expression data related to breast cancer tissue and adjacent tissue were downloaded from the TCGA database to screen the differentially expressed miRNAs in breast cancer and adjacent tissue. The GEO database was used to validate the expressions of miRNAs in 26 kinds of cell lines of TNBC and the changes in serum miRNAs in the TNBC patients before and after chemotherapy. The target gene function of candidate miRNAs was analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment and protein interaction network. Results:The TCGA database showed that the expression level of miR-21-5p in breast cancer tissue was significantly higher than that in adjacent tissue (logFC=5.557, P<0.01). The results of GEO database showed that the expression level of miR-21-5p increased in TNBC cell line was significantly higher; the relative expression levels in more than 20 kinds of cell lines from 26 TNBC cell lines were over 70 000, and the expression level of miR-21-5p in the TNBC patients after combined chemotherapy was significantly decreased(logFC=-5.07, P<0.01).The GO analysis showed that miR-21-5p played a regulatory role in DNA replication, transcription and vascular remodeling. The KEGG enrichment analysis showed that miR-21-5p mainly affected the occurrence and development of TNBC through mitogen activated protein kinase(MAPK) and transforming growth factor-β(TGF-β) pathways. Conclusion:miR-21-5p is up-regulated in TNBC tissue and plays a positive regulatory role in the progression of TNBC, which may be a key biomarker for identifying the prognostic extent of TNBC. DUSP8 may be involved in the regulation of the occurrence and development of TNBC as a target gene of miR-21-5p.

Objective:To investigate the distribution of JAK2V617F mutation in the patients with myeloproliferative neoplasms (MPN), to explore the association between JAK2V617F mutation and the clinical features of MPN, and to clarify its clinical significance in the diagnosis and treatment of the MPN patients. Methods:A total of 170 patients with MPN were selected as the subjects and divided into true polycythemia (PV) group (n=68), primary thrombocytopenia (ET) group (n=88) and primary myelofibrosis (PMF) group (n=14).The JAK2V617F mutations in 170 patients with MPN were detected by allele-specific PCR (AS-PCR). The differences in gender, age, white blood cell count, hemoglobin,platelet count, prothrombin time(PT),activated partial thromboplastin time(APTT), D-dimer, whether complicated with splenomegaly and thromboembolism of the JAK2V617F mutant-type and wild-type patients in each group were analyzed. Results:The total mutation rate of JAK2V617F mutation in 170 patients with MPN was 68.2% (116/170),and the mutation rates in PV, ET, and PMF groups were 72.1% (49/68), 68.1% (60/88), and 50.0% (7/14), respectively.The JAK2V617F mutation rate in PV group was higher than those in the other two groups, but there were no statistical differences(P=0.281). Compared with wild type group, the age of onset, the white blood cell and platelet counts of the PV patients with JAK2V617F mutation were significantly increased(P<0.01), PT and APTT were significantly prolonged(P<0.01), and the incidence of splenomegaly was increased (P<0.05); the white blood cell count of the the ET patients with JAK2V617F mutation was increased(P<0.01),the hemoglobin level was decreased(P<0.01), APTT was significantly prolonged(P<0.01), and the incidence of thromboembolic events was increased (P<0.05); the age of onset, the white blood cell and platelet counts of the PMF patients with JAK2V617F mutation were signficantly increased (P<0.05 or P<0.01).Compared with the ET and PMF patients with JAK2V617F mutation, APTT in the PV patients with JAK2V617F mutation was significantly prolonged(P<0.05). Compared with the JAK2V617F mution PV and ET patients, the age of onset and white blood cell count of the PMF patients with JAK2V617F mutation were increased(P<0.05). Conclusion:The clinical characteristics of the MPN patients with JAK2V617F mutation are significantly different from those in the wild-type patients. The clinical features of PV, ET and PMF in the patients with JAK2V617F mutation are also different. The PV patients with JAK2V617F mutation are more prone to APTT prolongation. The PMF patients with JAK2V617F mutation have the higher onset age and the white blood cell count.

Objective:To study the vitamin D levels and deficiency rates in the patients with chronic kidney disease (CKD) in different stages, and to clarify the value of serum vitamin D levels in the evaluation on nutritional status of the patients with CKD. Methods:The patients diagnosed as CKD in the Department of Nephrology and volunteered to participate in the survey were selected as the subjects. According to the different renal function stages, the patients were divided into early stage (CKD1-2 stage) group(n=115),middle and late stage(CKD3-5 stage) group(n=131) and dialysis group(n=38); the serum 25(OH) D levels and vitamin D deficiency rates of the patients in various groups were compared. According to K/DOQI guidelines, the patients were divided into vitamin D deficiency group(n=196), vitamin D insufficiency group(n=76), and vitamin D sufficiency group(n=12); the red blood cell (RBC) count, hemoglobin (Hb), hematocrit (Hct), mean red blood cell volume (MCV), mean red blood cell hemoglobin (MCH), mean red blood cell hemoglobin concentration (MCHC), serum albumin (Alb), prealbumin (PA), total protein (TP), creatinine (Cr), blood urea nitrogen (BUN), 25 hydroxy vitamin D[25(OH)D] and urea protein (Up) of the patients in various groups were compared. The serum 25(OH) D-related factors were analyzed by Spearman correlation analysis method; the influencing factors of serum 25 (OH) D level were analyzed by multiple linear regression analysis. Results:The vitamin D deficiency rates of the patients in early stage group, middle and late stage group, and dialysis group were 77.4%, 66.4%, and 52.6%, respectively; the serum 25(OH) D level of the patients in early stage group was significantly lower than those of the patients in middle and late group and dialysis group (P<0.05); the vitamin D deficiency rate of the patients in early stage group was significantly higher than that of the patients in dialysis group (P<0.05).The count of RBC,the level of Hb,and Hct of the patients in vitamin D deficiency group were significantly lower than those of the patients in Vitamin D insufficiency group (P<0.05).The Cr levels of the patients in vitamin D deficiency group and vitamin D insufficiency group were significantly lower than that in vitamin D sufficiency group (P<0.05).The levels of Alb and TP of the patients in vitamin D deficiency group were significantly lower than those in other two groups (P<0.05); the level of Up in vitamin D deficiency group was significantly higher than that in vitamin D deficiency group (P<0.05).The Spearman correlation analysis results showed that the serum 25(OH) D level was positively correlated with the RBC count, Hb level, Hct, MCV, Cr, PA, Alb,and TP levels(r=0.199,P=0.01;r=0.232,P<0.01;r=0.232,P<0.01;r=0.131,P=0.028;r=0.147,P=0.013;r=0.277,P<0.01;r=0.696,P<0.01;r=0.677,P<0.01); and it was negatively correlated with Up (r=-0.603, P<0.01).When 25 (OH) D was selected as the dependent variable,the RBC count, Hb, Hct, MCV, PA, Alb, TP, Cr, and Up as the independent variables, multiple linear regression analysis was performed; as Hb was positively correlated with Hct and the RBC count(r=0.974, P<0.01; r=0.943, P<0.01) and Alb was positively correlated with TP (r=0.874, P<0.01), Hb, MCV, PA, Alb, Cr,and Up were selected as the independent variables in order to avoid collinearity. The multiple linear regression analysis results showed that Alb and Up were the independent factors of 25(OH) D level. Conclusion:Vitamin D deficiency is a serious problem in the patients with CKD in Dalian area, especially in the patients with early stage CKD. The too low serum 25(OH)D levels are associated with anemia, hypoproteinemia, and urinary protein loss in the patients with CKD.

Objective:To explore the influencing factors related to the quality of life of the patients with idiopathic interstitial pneumonia (ⅡP), and to provide the theoretical basis for improving the quality of life, preventing other complications,and improving the prognosis of the patients with ⅡP. Methods:St George's Respiratory Questionnaire (SGRQ) was used to evaluate the quality of life of 300 hospitalization patients with ⅡP who met the inclusion criteria in the study. The differences of quality of life among different groups of people were compared. A general linear regression model was used to screen the independent influencing factors that may affect the scores of quality life of all dimensions. Results:Drinking, forced vital capacity (FVC) and 6 min walk test (6MWT) were the independent influencing factors of the symptom score of the ⅡP patients (P<0.05); nation, family per capita monthly income, carbon monoxide diffusion capacity (DLCO), and 6MWT were the independent influencing factors of activity limit score of the ⅡP patients (P<0.05); nation, family per capita monthly income, FVC, and 6MWT were the independent influencing factors of life impact score of the ⅡP patients (P<0.05); nation, family per capita monthly income, FVC, and 6MWT were the independent influencing factors of the scores of quality of life of the ⅡP patients (P<0.05). Conclusion:The activity limit of ⅡP patients is more obvious. Drinking, nation and family per capita monthly income, FVC, 6MWT and DLOC are the factors affecting the quality of life of the ⅡP patients in different dimensions.

Objective:To study the therapeutic effect of compound Chinese medicine preparation of wild chrysanthemum and rhizoma drynariae combined with ornidazole in the treatment of chronic periodontitis,to explore the curative effect of this method in the treatment of chronic periodontitis,and to calarify its mechanism. Methods:A total of 112 patients with chronic periodontitis were divided into control group, ornidazole group, Chinese medicine group, Chinese medicine combined with ornidazole group (combination group) according the order of clinic time;there were 28 cases in each group. After basic periodontal treatment of the affected tooth(supragingival scaling and subgingival scaling), the periodontal pockets of the patients in control group were washed with 3%H₂O₂ and 0.9%NaCl injection by turns,once a week; the patients in ornidazole group were orally administered with ornidazole one piece at a time, twice a day;the patients in Chinese medicine group were injected with compound Chinese medicine preparation of wild chrysanthemum and rhizoma drynariae into the pocket to make the medicine overflow to the edge of the gingival margin,once a week; the patients in combination group were injected with compound Chinese medicine preparation into the periodontal pocket while orally administered with ornidazole. All patients in various groups were treated for 4 weeks.The periodontal indexes including modified plaque index(mPLI), modified sulcus bleeding index (mSBI),probing depth(PD), and attachment loss (AL) of the patients in various groups were observed before and after treatment; and the levels of tumor necrosis factor-α(TNF-α) and prostaglandin E₂ (PGE₂) in the gingival crevicular fluid (PISF) of the patients in various groups were detected after one course of treatment. Results:There were no significant differences in the periodontal indexes and the levels of TNF-αand PGE₂ in PISF of the patients before treatment between various groups(P>0.05).Compared with control group,the level of PGE₂ in PISF in Chinese medicine group was reduced(P<0.05);the PD and the PGE₂ level in PISF of the patients in ornidazole group were significantly reduced (P<0.05);the mPLI,mSBI,PD and AL, the levels of TNF-α and PGE₂ in PISF of the patients in combination group were significantly reduced (P<0.05).Compared with ornidazole group, there were no significant differences in the above indicators of the patients in Chinese medicine group (P>0.05);the mPLI, mSBI, PD and AL,the TNF-α level in PISF of the patients in combination group were significantly reduced (P<0.05).Compared with Chinese medicine group,the mPLI, mSBI, PD and AL,the levels of TNF-α and PGE₂ in PISF of the patients in combination group were significantly reduced(P<0.05). Conclusion:The traditional compound Chinese medicine preparation of wild chrysanthemum and rhizoma drynariae combined with ornidazole has the best therapeutic effect in the chronic periodontitis,which can improve the periodontal index and reduce the levels of TNF-α and PGE₂ in PISF of the patients.

Objective:To explore the intervention effects of aerobic exercise(AE) and AEcombined with resistance training(AE+RT) on the body composition, cardiovascular function and the serum C-reactive protein (CRP) level in the male obese college students, and to provide the evidence for exercise prescription in the male obese college students. Methods:A total of 36 male obese college students were chosen and randomly assigned into control group, AE group and AE+RT group(n=12). The students in AE and AE+RT groups conducted a 12-week (5 times per week and 60 min per time) exercise protocols and the students in control group did not perform regular physical training during 12 weeks of study. Then the body weight(BW),body mass index(BMI),fat mass(FM), percent of body fat(BF%), muscle mass(MM), waist circumference(WC) and the serum CRP levels of the subjects were measured before and after exercise;the cardiac function including heart rate(HR), stroke volume(SV) and cardiac output(CO), vascular function including mean systolic pressure(MSP), mean diastolic pressure(MDP), vascular elastic dilatation coefficient(VDC) and total cycle resistance(TCR) and blood status (V) were evaluated by cardiovascular function tester. Results:After 12-week training, BW, BMI, FM, and BF% of the students in AE and AE+RT groups were significantly decreased (P<0.05 or P<0.01) compared with before exercise, MM in AE+RT group was significantly increased(P<0.01) and WC was significantly decreased (P<0.01). After 12-week training, HR, MSP, TCR, and V in AE and AE+RT groups were significantly decreased (P<0.05 or P<0.01), SV and VDC were increased (P<0.05 or P<0.01); MDP,CO,and the serum CRP level in AE group had no significant differences between before and after exercise,and MDP and the serum CRP level after exercise in AE+RT group were significantly decreased (P<0.01), CO was increased (P<0.01) compared with before exercise. Compared with AE group, the FM, BF%, HR, MSP, TCR, and the serum CRP level of the students in AE+RT group after 12-week exercise were significantly decreased (P<0.05 or P<0.01); MM, SV, and CO were significantly increased (P<0.05 or P<0.01); but there were no significant differences (P>0.05) in WC, BMI, MDP, VDC, and V between AE and AE+RT groups. Conclusion:AE and AE+RT can improve the body composition and cardiovascular function of the male obese college students, and AE+RT is superior to single AE, while AE+RT can decrease the serum CRP level.

Objective:To explore the curative effect of N-acetylcysteine (NAC)inhalation in the treatment of the elderly patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD), and to clarify the application value of NAC in the regular treatment of the AECOPD patients. Methods:A total of 78 elderly patients with AECOPD were enrolled and randomly divided into control group(n=38) and NAC group(n=40). The patients in two groups all received regular treatment; the patients in NAC group received additional NAC inhalation (0.3 g, 2 times per day) while the patients in control group received normal saline inhalation. The arterial partial pressure of oxygen (PaO₂), arterial partial pressure of carbon dioxide (PaCO₂), forced expiratory volume in 1 second(FEV1), forced expiratory volume in FEV1% predicted(FEV1%pred), the clinical COPD Questionnaire(CCQ) score, as well as the rates of intravenous steroid infusion, the incidence of hospital infection and the duration of hospitalization before and after treatment were recorded. Results:Compared with control group, the difference values of PaO₂ of the patients in NAC group was significantly increased(P=0.033),but there were no significant differences in the difference values of PaO₂,FEV1 and FEV1% pred(P>0.05);the CCQ score of the patients in NAC group was significantly higher than that in control group,and there was significant difference in the difference values(P<0.05).There were no significant differences in the rates of intravenous steroid infusion,the incidence of hospital infection and the duration of hospitalization of the patients between two groups(P>0.05). Conclusion:NAC inhalation could apparently improve the PaO₂ and the clinical symptoms in the elderly AECOPD patients, but has no significant effects on the pulmonary function, the rate of intravenous steroid infusion,the incidence of hospital infection and the duration of hospitalization.

Objective:To observe the curative effect of non-surgical treatment under periodontal endoscope in the patients with moderate-to-severe periodontal pockets of multirooted teeth after scaling and root planning(SRP), and to expound the clinical significance of periodontal endoscope in assisting periodontal non-surgical treatment. Methods:After SRP, there were still more than 127 multirooted teeth with moderate-to-severe periodontal pockets in 24 patients with periodontitis. The above multirooted teeth received non-surgical treatment under periodontal endoscope, the control of periodontal risk factors was evaluated by the periodontal risk assessment(PRA) model; the probing depth(PD),bleeding on probing (BOP), attachment loss (AL), calculus residual rate of the patients after treatment were detected by Florida Probe system; the percentage of furcation defect was detected. Results:After 3 months of periodontal endoscope-assisted periodontal non-surgical treatment, the risk factors of the patients after treatment were reduced and the patients had good prognosis.The PD value and percentage of BOP positive sites of the teeth of the patients after treatment were significantly decreased compared with before treatment(P<0.01); the PD value of the near-distal median point was obviously decreased compared with the PD value of the central site(P<0.01);the positive rate of calculus was significantly decreased(P<0.01);there was no significant difference in the AL(P>0.05). The percentage of teeth with Class Ⅰ furcation involvement among the multirooted teeth after treatment was significantly lower than before treatment (P<0.05), but the percentage of teeth with Class Ⅱ and above furcation involvement had no significantly difference between before and after treatment (P>0.05). Conclusion:The effect of periodontal endoscope-assisted non-surgical treatment for the multirooted teeth with residual moderate-to-sever periodontal pockets after SRP, and it can avoid periodontal operation to some extent.

Objective:To observe the clinical efficacy and safety of apatinib combined with chemotherapy in the patients with advanced breast cancer after failed multi-line therapy, and to clarify the siginificance of apatinib in the treatment of the patients with advanced breast cancer. Methods:Twenty-five patients with advanced breast cancer were treated with multi-line therapy,among them 5 (20%) patients were in the third-line treatment, 7 (28%) patients were in the fourth-line treatment, and 13 (52%) patients were in the fifth-line treatment and above. All patients were treated with apatinib in combination with chemotherapy, and the chemotherapy regimen was selected based on the condition and previous medication. Apatinib 250-500 mg was given orally once a day, until the disease progresses occured or the patients could not tolerate the adverse reactions. The efficacies, including the objective response rate(ORR), the clinical benefit rate(CBR), and progression-free survival (PFS), were evaluated by RECIST 1.1. The adverse reactions were evaluated by NCI-CTC 4.0. Results:The median number of treatment lines of the patients with breast cancer was fifth-line, the total ORR was 12% (3/25), the CBR was 52% (18/25), and the median progression-free survival (mPFS) was 6.00 months. Among the 5 patients with third-line therapy, 2 patients were stable disease(SD) and the CBR was 40% (2/5); among the 7 patients received the fourth-line therapy, 2 patients were partial response(PR), 2 patients were SD, and the CBR was 57%(4/7); among the 13 patients received firth-line therapy, 1 patient was PR, 6 cases were SD, and the CBR was 54% (7/13). According to the pathological type, among 5 patients of triple-negative type, 3 patients were SD, the CBR was 60% (3/5); among 12 patients of Luminal type,1 patients was PR, 2 patients were SD, and the CBR was 25% (3/12); among 8 patients of HER-2 positive type, there were 2 patients acheived PR, 5 patients acheived SD, and the CBR was 88% (7/8). The short-term efficacy of the patients in 50 years old and over group was better than that of the patients in below 50 years old group (P<0.05). There were no significant differences in the short-term efficacies between the other factors (P>0.05). Moreover, the patients treated with apatinib combined with chemotherapy had good tolerance, and the main adverse reactions were fatigue, hand-foot syndrome, hepatic insufficiency, hypoproteinemia, anemia, anepi-thymia,hypertension,and proteinuria;mainly in grade 1 or grade 2;the most common adverse reactionswere fatigue(80%),hypoproteinemia(60%),hand-foot syndrome(60%),and hepatic insufficiency(60%). Conclusion:Apatinib mesylate combined with chemotherapy is effective in the treatment of the advanced breast cancer patients failed in multi-line therapy and the patients can tolerate the adverse reactions.

Objective:To analyze the curative effect of phacoemulsification and to intraocular lens implantation combined with anterior vitrectomy in the children with congenital cataract, and to investigate its effectiveness and security. Methods:A total of 43 cases(49 eyes) of 3-7 years old congenital cataract children who were admitted to our hospital were selected as the subjects. According to different treatment methods, the children were divided into control group (23 cases with 26 eyes) and treatment group (20 cases with 23 eyes). The children in control group were treated with phacoemulsification and intraocular lens implantation and the children in treatment group were treated with phacoemulsification, intraocular lens implantation and anterior vitrectomy. After operation, the vision of the children in two groups was observed; the intraocular pressure(IOP), anterior chamber depth, posterior capsule turbidity and the complications in operation and after operation of the children in two groups were detected by non-contact tonometer and ultrasonic biological microscope. Results:The children in two groups were followed up for an average of 12 months after surgery,and 49 eyes were follow-up after operation.Compared with control group, the vision of the children in treatment group was improved significantly,and the constituent ratio of the children with corrected visual acuity after opertion ≥ 0.5 was significantly increased(P<0.05);the anterior chamber depths was significantly increased(P<0.05) and the posterior capsular opacity was significantly reduced(P<0.05). There were no significant differences in the incidences of IOP, corneal endothelium edema(CE), anterior chamber flash(ACF), and anterior chamber cellulosic exudation(ACCE) of the children in two groups(P>0.05). Conclusion:Phacoemulsification and intraocular lens implantation combined with anterior vitrectomy in the children with congenital cataract can effectively improve the visual acuity, anterior chamber depth and posterior capsule opacity; it is more helpful for the reconstruction of visual function after operation in the children.

Objective:To investigate the clinical symptom improvement time of the children with non-diarrhea-related hemolytic uremic syndrome(HUS) treated with glucocorticoids,and to evaluate its efficacy. Methods:A total of 22 children with non-diarrhea-related HUS were selected as the subjects. According to whether glucocorticoid was used in combination of plasma in the actue stage of the children, the children were divided into simple plasma group (n=11) and plasma combined with glucocorticoid treatment group (n=11). The average hospital stays, the time of hematuria, the time of platelets and serum creatinine to recover to the normal levels of the children in two groups were analyzed. The clinical material of children in two groups after hospital discharge were collected,and the prognosis of the children in two groups was evaluated according to the follow-up results. Results:Compared with simple plasma group, the average hospital stays,and the time of hematuria and the time of serum creatinine to recover to the normal level of the children in plasma combined with glucocorticoid treatment group were shortened,but there were no significant differences(P>0.05). The time of platelets rising to the normal level was similar had no significant difference between two groups(P>0.05). Ten cases completed the follow-up in simple plasma group;one children suffered hypertension during follow-up,and one case replapsed 1 year after operation.In plasma combined with glucocorticoid treatment group, nine cases completed the follow-up;one children persistented urinalysis abnormalities and hearing loss,with the growth and development being slower than their peers;the remaining children all achieved the clinical cure standards. The clinical cure rate of the chidren in plasma combined with glucocorticoid treatment group was 88.9%,while it was 80.0% in simple plasma group; there was no significant difference in the clinical cure rate between two groups(P>0.05). Conclusion:Using glucocorticoids in the acute stage of non-diarrhea-related hemolytic uremic syndrome in the children could not shorten the acute course and improve the prognosis of the children.

Objective:To analyze the clinical features of the patient with myelodysplastic syndrome who developed bilateral ischemia necrosis of femoral head after allogeneic hematopoietic stem cell transplantation,and to discuss the perioperative management, efficacy and prognosis of bilateral hip arthroplasty for such patients. Methods:The clinical material of one patient with myelodysplastic syndrome who developed bilateral ischemia necrosis of femoral head after allogeneic hematopoietic stem cell transplantation including age, gender, clinical symptoms, previous medical history and outcomes, auxiliary examinations, treatment regimen and prognosis were collected, and the relevant literatures were reviewed. Results:After allogeneic hematopoietic stem cell transplantation, the patient was diagnosed with bilateral ischemia necrosis of femoral head (Ficat stage:left stage Ⅳ, right stage Ⅲ);the patient presented bilateral hip joint pain and severe limitation of movement. Admission to complete preoperative examination, through multiple consultations of Department of Hematology (blood routine, coagulation routine, erythrocyte sedimentation rate, cyclosporine concentration) and Department of Anesthesiology (blood routine, coagulation routine, liver and kidney function, cardiopulmonary function and lower extremity venous color Doppler), bilateral total hip arthroplasty was decided to perform. The operation went on smoothly,the patient recovered quickly after operation, and the symptoms of pain were improved significantly. The first day after operation, the patient went to the ground, and walked with two crutches. There was no complications during perioperative period and postoperation. One month after operation, the patient recovered well,blood routine examination, liver function, renal function, erythrocyte sedimentation rate, C-reactive protein and pelvic positivity were all normal. Conclusion:Total hip arthroplasty is effective in the patients with bilateral ischemia necrosis of femoral head after allogeneic hematopoietic stem cell transplantation. Strengthening perioperative management can effectively prevent the complications and improve the quality of life of the patients.

Objective:To explore the process of diagnosis and treatment of the patients with dens invaginatus of right maxillary second molar, to analyze the clinical features, the radiographic features and the pathological features of the dens invaginatus, and to make a reasonable treatment plan. Methods:The clinical data, imaging findings and pathological data of a patient with dens invagination were collected. The clinical diagnostic criteria and treatment methods of these cases were summarized with relevant literature review. Results:The patient, female, 28 years old, was diagnosed with a hole in the upper right posterior teeth for 6 months. The special examination results showed that the 17-toothed face was flat, the crown was huge, and the occlusal surface was seen in the sag and sputum; the medullary cavity was not reached. The radiograghic examination results showed that the enamel density image of the 17-toothed surface was sunken to the root, with a depth of about 7.7 mm, diagnosed as dens invaginatus. At the time of initial treatment, the humus was removed and the sag received sedative treatment. The spontaneous pain still existed during the follow-up examination, and the problem of food embolism was not solved. Considering the poor recovery after treatment, the patient agreed to remove the tooth. The isolated tooth was observed by pathological grinding, and the enamel and dentin structures of the invaginatus were abnormal observed under microscope. Conclusion:The patients with dens invagination occurs in the molar area are rare;the type of disease and treatment plan should be determined by imaging examination during the process of diagnosis and treatment; it suggests that the patients with poor recovery after treatment should remove the teeth.

Objective:To explore the effects of two kinds of calcium phosphate transfection methods in the 293T cells, and to establish the method of achieving high-efficiency and stable calcium phosphate transfection in the 293T cells. Methods:Fluorescence microscope was used to observe the transfection efficiencies of transfection of pCDH-GFP-3xflag-TRAF6 plasmid into the 293T cells by two kinds of calcium phosphate transfection methods (traditional calcium phosphate transfection method and improved calcium phosphate transfection method). Real-time PCR and Western blotting method were used to detect the expression levels of TRAF6 mRNA and flag protein in the 293T cells after transfection of pCDH-GFP-3xflag-TRAF6 plasmid by two kinds of calcium phosphate transfection methods. Results:Under fluorescence microscope, compared with traditional calcium phosphate transfection method, the transfection efficiencies of improved calcium phosphate transfection method 24 an 48 h after transfection of pCDH-GFP-3xflag-TRAF6 plasmid into the 293T cells were significantly increased (P<0.01). The Real-time PCR and Western blotting results showed that compared with traditional transfection method, the expression levels of TRAF6 mRNA and flag protein in the 293T cells 24 and 48 h after transfection of pCDH-GFP-3xflag-TRAF6 plasmid by calcicum phosphate transfection method were significantly increased(P<0.01). Conclusion:The improved calcium phosphate transfection method established in this reseach is a highly efficient and stable DNA transfection method.