Objective:To explore the effects of repeated transcranial direct current stimulation (tDCS) on the depression behaviors in the rat models of Parkinson' s disease (PD),and to clarify the neurochemical mechanism of tDCS in affecting the PD-related depression behaviors. Methods:A total of 32 rats were randomly divided into sham operation group,PD group,anodal repeated tDCS group and cathodal repeated tDCS group,8 rats in each group.Except sham operation group,the rats in other groups were used to establish the PD models.The depression behaviors immobility time and sucrose consumption of the rats in various groups were observed by forced swimming test and sucrose preference test.The monoamine levels in medial prefrontal cortex (mPFC) and raphe nuclei (RN) of the rats in various groups were assessed by HPLC-electrochemistry method. Results: Compared with PD group,the immobility time in anodal repeated tDCS group and cathodal repeated group was significantly decreased (P<0.05 or P<0.01);the sucrose consumption in anodal repeated tDCS group was significantly increased (P<0.05),but no significant change was observed in cathodal repeated tDCS group (P>0.05).Compared with PD group,the levels of dopamine (DA) and 5-hydroxytryptamine (5-HT) in mPFC and RN of the rats in anodal repeated tDCS group were significantly increased(mPFC:P<0.05 or P <0.01; RN:P<0.01); the level of 5-HT in mPFC of the rats in cathodal repeated tDCS group was significantly increased (P<0.05). Conclusion:The repeated tDCS,especially the anodal repeated tDCS,can alleviate the depression behaviors in the rat models of PD,and their mechanisms may be related to the increasing of the monoamine levels in the related brain regions.

Objective:To explore the protective effect of overexpression of uncoupling protein 2 (UCP2) on the mitochondrial in cardiomyocytes of the rats with sepsis, and to clarify its mechanisms. Methods:The H9C2 cardiomyocytes with UCP2 overexpression were reconstructed and randomly devided into normal control group, lipopolysaccharide/peptidoglycan(LPS/PepG) group,PHBLV group and PHBLV-UCP2 group.The H9C2 cells in various groups except normal control group were stimulated with LPS/PepG.The expression levels of UCP2 mRNA and protein in the cardiomyocytes in various groups were detected with RT-PCR and Western blotting methods.The levels of creatine kinase (CK) and tumor necrosis factorα(TNF-α) were measured by spectrophotometer and enzyme-linked immunosorbent assay (ELISA).The mitochondrial morphology of cardiomyocytes in various groups was observed by transmission electron microscope (TEM),the mitochondrial membrane potential (MMP) levels of cardiomyocytes in various groups were detected by flow cytometry (FCM),and the levels of reactive oxygen species (ROS) and adenosine triphosphate (ATP) in mitochondria of cardiomyocytes were measured by multimode reader. Results:Compared with normal control group,the levels of CK and TNF-α in LPS/PepG group were increased significantly(P<0.05),the mitochondrial morphology was injured obviously,the MMP level was decreased (P<0.05),the level of ROS was increased (P<0.05), and the ATP level was decreased (P<0.05).Compared with LPS/PepG group,the CK and TNF-α levels in cardiomyocytes in PHBLV-UCP2 group were decreased (P<0.05),the injury of mitochondria was improved,the MMP level was increased (P<0.05),the ROS level was decreased signficantly(P<0.05),and the ATP level was significantly increased(P<0.05).The levels of CK,TNF-α,MMP,ROS,ATP in the cardiomyocytes and the degree of mitochondrial swelling in PHBLV group had no obovionsly changes compared with LPS/PepG group (P>0.05). Conclusion: UCP2 overexpression plays a protective role in myocardial mitochondria of the rats with sepsis,which may be related to its regulation on the membrane potential to affect the mitochondrial ROS and ATP synthesis through uncoupling effect under septic condiction.

Objective:To study the effects of starvation on the autophagy and apoptosis of Ana-1 cells,and to explore their mechanisms. Methods:The Ana-1 cells were cultured in vitro and starved for 0 h(control group),3,6,9,12 and 24 h(starvation group)to study the effects of starvation on the autophagy and apoptosis.The Ana-1 cells were divided into blank control group,starvation group,3-methyladenine group(3-MA) and 3-MAcombined with starvation group,and they were incubated for 24 h to explore their mechanisms.The ratio of autophagy associated protein LC3-Ⅱ/LC3-Ⅰ,the expression levels of Beclin-1 and apoptosis-related protein Caspase-3 in the cells in various groups were detected by Western blotting method.Cell morphology 6,12,and 24 h after starvation was observed by inverted microscope. Results:Compared with control group,the ratio of LC3-Ⅱ/LC3-Ⅰ in the Ana-1 cells in starvation group(6,12,and 24 h)and the expression levels of Beclin-1 in starvation group(3,6,9,12,and 24 h) were significantly increased(P<0.05 or P<0.01),and the Caspase-3 levels in starvation group(3,6,9,12,and 24 h) were decreased significantly(P<0.05 or P<0.01) in a time-dependent manner.Compared with control group,the cells in starvation group at different time were changed in shape:the cells shrank,the fragments appeared,and the changes of cells became more obvious with the prolongation of time.Compared with starvation group,the ratio of LC3-Ⅱ/LC3-Ⅰ and the expression levels of Beclin-1 and Caspase-3 in the Ana-1 cells in 3-MA combined with starvation group were decreased significantly(P<0.05 or P<0.01). Conclusion:Starvation could induce the autophagy and apoptosis of the Beclin-1-dependent Ana-1 cells.

Objective:To investigate the effects of Dendrobium candidum (DC)on the insulin resistance in the MKR mice and the MIN6 cells, and to illuminate the possible mechanisms. Methods:The MKR mice aged 9 weeks were randomly divided into 4 groups,and the mice in various groups were treated with 10,20,40 g·kg^-1 DC and normal saline(blank control group) by gavage,respectively,for 6 weeks.The blood glucose levels of the mice in various groups were detected bi-weekly,and the optimum concentration of DC in reducing the glucose level.The male MKR mice aged 8 weeks were randomly divided into control group,DC group,Met group and Met+DC group.The mice were treated with normal saline and the corresponding drugs for 8 weeks,and the blood glucose levels of the mice were determined.Glucose tolerance test(GTT) and insulin tolerance test(ITT) were performed after administration.The qPCR method was used to detect the expression levels of genes related to glucose and lipid metabolism in liver and subcutaneous fat tissues of the mice.The MIN6 cells cultured in vitro were divided into control group,high concentration of palmitic acid(H-PA) group,high concentration of glucose (H-Glu) group,H-PA+H-Glu group,H-PA+DC group,H-Glu+DC group,and H-Glu+H-PA+DC group.MTT method and ELISA method were used to detect the proliferation activities and insulin secretion levels of cells in various groups.Flow cytometry was used to measure the apoptotic rates in various groups.Western blotting method was used to detect the protein expression levels of PI3K/AKT/signaling pathway in the cells in various groups. Results:In vivo,the optimum concentration of DC in reducing the blood glucose level was 20 g·kg^-1·d^-1.Compared with control group,the blood glucose level of the mice in DC,Met and Met+DC groups were decreased significantly (P<0.05);compared with DC group and Met group,the blood glucose level of the mice in Met+DC group was decreased (P<0.05). Compared with DC group and Met group,the glucose tolerance and insulin sensitivity were increased,and the insulin resisitance was improved.Compared with control group,the Pepck mRNA expression level in liver tissue of the mice in Met+DC group was decreased significantly(P<0.05),and the GCK mRNA expression level was increased significantly (P<0.05);the Ppar-γ mRNA expression level in subcutaneons fat tissue of the mice in Met group and Met+DC group were increased significantly(P<0.05).In the MIN6 cells,160 μmol·L^-1and 30 μmol·L^-1 were confirmed to use as the end concentrations of H-PA and H-Glu to induce the insulin resistance,and the cells in various groups were treated with 1.44 g·L^-1 DC.Compared with H-PA group,H-Glu group and H-PA+H-Glu group,the insulin secretion levels of MIN6 cells in H-PA-DC group,H-Glu+DC group and H-PA+H-Glu+DC group were increased significantly(P<0.05),the apoptotic rates were decreased,and the expression levels of p-IRSp-1, p-PI3K and p-AKT proteins was increased significantly(P<0.05);the expression level of p-GSK3β protein in H-PA+H-Glu+DC group was significantly decreased(P<0.05). Conclusion: DC can ameliorate the insulin resistance in the MKR mice. The effect of hypoglycemic therapy is more obvious with metformin in combination. In vitro, DC can improve the palmitic acid and glucose-induced insulin resistance through the PI3K/AKT signaling pathway and increase the insulin secretion level and reduce the apoptotic rates.

Objective: To construct the recombinant expression vector of mitochondria-targeted KillerRed (KR), and to explore the producing regularity of reactive oxygen species (ROS) induced by visible light exposure, and its enhancement in the proliferation inhibition of radiation on the HeLa cells. Methods: Gene recombination technique was used to build the recombinant vectors plxsp-flag-Sarm1-KR and plxsp-flag-Sarm1-mCherry targeting mitochondria. The cervical cancer HeLa cells were divided into control, plxsp-flag, plxsp-flag-Sarm1-KR, 4 Gy, plxsp-flag + 4 Gy and plxsp-flag-Sarm1-KR + 4 Gy groups. After the vectors were transfected into the cells for 24 h, the expression levels of mCherry and mitochondrial track COXⅣ proteins were determined by fluorescence microscope;DCFH-DA probe was used to detect the mean fluorescence intensity(MFI) to indicate the ROS producing level. After 4 Gy X-ray irradiation,the cell proliferation activity was measured by CCK8 kits. Results: After the vectors were sequenced and identified, the sequence was consistent with that in GenBank, and the results showed the fusion expression vector plxsp-flag-Sarm1-KR(mCherry) was successfully constructed. After the plasmids were transfected into the HeLa cells, under fluorescence microscope, mCherry protein and COXⅣ protein had the same expression location. From ROS producing level, in control and plxsp-flag groups, after the cells were exposed to visble light for 10, 30 and 60 min, the MFI had no obvious changes after the probe was incorporated for 10, 30 and 60 min; but in plxsp-flag-Sarm1-KR group, the MFIs were increased with the time prolongation; compared with control group,the cells were exposed to visble light for 10 and 30 min, and the MFIs were significantly increased after the probe was incorporated for 60 min (P<0.05); when the cells were exposed to visble light for 60 min, the MFIs were significantly decreased after the probe was incorporated for 30 and 60 min (P<0.05). Compared with control group, the A(450) in plxsp-flag group had no obvious change(P>0.05); the proliferation activities were significantly decreased at 10 and 24 h in plxsp-flag-Sarm1-KR group (P<0.05); the proliferation activities were significantly decreased at 10 h in 4 Gy and plxsp-flag + 4 Gy groups (P<0.01); the proliferation activities were significantly decreased at 10, 24 and 48 h in plxsp-flag-Sarm1-KR + 4 Gy group (P<0.05 or P<0.01). The proliferation activity in plxsp-flag-Sarm1-KR + 4 Gy group was lower than those in plxsp-flag-Sarm1-KR and 4 Gy groups (P<0.05). Conclusion:The mitochondria-targeted fusion expression vectors are successfully constructed,and the KR proteins can induce the ROS producing and enhance the inhibitory effect of proliferation of ionizing radiation on the HeLa cells.

Objective: To screen the active constituents of pueraria against hyperlipoproteinemia and predict the potential targets and signal pathways, and to investigate the therapeutic mechanism based on network pharmacology. Methods: All the chemical constituents related to pueraria form the TCM Database were collected.The drugs with the similar structures of chemical constituents from pueraria were screened and the targets were predicted using Drugbank database.The proteins related to hyperlipoproteinemia were found using OMIM database.Anti-hyperlipoproteinemia target-functionally related protein interaction of puerariae network and active constituents of pueraria-target-functionally related protein network were constructed using STRING database and software Cytoscape 3.6.0;and topological analysis and clustering analysis were performed to predict the crucial proteins of pueraria in the treatment of anti-hyperlipoproteinemia.Pathway enrichment analysis was performed in the validated targets to predict the pathways of Pueraria through the Kyoto Encyclopedia of genes and genomes (KEGG). Results:4'-Methoxy puerarin,arachidic acid,daidzein 4',7-diglucoside,daidzin,dauricine and methyl-p-hydroxycinnamate were the main 7 kinds of active constituents of pueraria.There were 20 potential targets of pueraria in treatment of hyperlipoproteinemia,including proto-oncogene tyrosine-protein kinase(Src),nuclear factor KB(NF-KB),peroxisome prolifeator-activated receptors(PPARs),estrogen receptor(Esr1),aryl hydrocarbon receptor(AHR),apolipoprotein A(APOAⅠ,APOAⅡ,APOAⅤ), apolipoprotein B (APOB),apolipoprotein C (APOCⅠ and APOCⅡ),apolipoprotein E (APOE),low density lipoprotein receptor (LDLR),lipoprotein lipase (LPL),ATP-binding cassette transporter A1 (ABCA1),lysophosphatidic acid A (LPA),glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1),fibrinogen A (FGA),cytochrome P450 (Cyp158A2) and androgen receptor (AR).In Kyoto encyclopedia of genes and genomes(KEGG) pathway enrichment analysis,there were 8 KEGG pathways,mainly involving PPAR system. Conclusion: Seven pharmacodynamic molecules of hyperlipoproteinemia treated with pueraria are screened by network pharmacology.20 crucial targets and 8 related pathways are obtained through the analysis of the active ingredient-target-disease network.Pueraria may regulate the cholesterol,triglyceride and low-density lipoprotein (LDL) metabolism through the PPARs,AR,LDLR and other receptors and related proteins in the treatment of hyperlipoproteinemia.

Objective:To investigate the estrogenic effects of BPA and DES co-administered or administered single in the immature female SD rats,and to provide the scientific evidences for further study on the toxicological mechanism of environmental estrogens(EEs). Methods:The immature female SD rats (21 d) were administered with BPA (200,400, and 800 mg·kg^-1),DES (15 and 150 μg·kg^-1) and the mixture of BPA and DES (100 mg·kg^-1 BPA + 7.5 μg·kg^-1 DES,200 mg·kg^-1 BPA + 7.5 μg·kg^-1 DES,400 mg·kg-1 BPA+7.5 μg·kg^-1DES,400 mg·kg^-1 BPA + 75 μg·kg^-1 DES); at the same time,control group was set up. The uterine weights,the ratios of uterine weights to body weights,the heights of luminal epithelium,the thickness of myometrium,the amounts of uterine glands,and the amounts of mature follicle of the rats in various groups were observed. Results:The uterine weight,the ratio of uterine weight to body weight,the height of luminal epithelium,the thickness of myometrium,and the amount of uterine glands of the rats in 800 mg·kg^-1 BPA group were increased significantly compared with control group (P<0.05).The uterine weights,the ratios of uterine weights to body weights,the heights of luminal epithelium,the thickness of myometrium,the amount of uterine glands and the amounts of mature follicle in 15 and 150 μg·kg^-1 DES groups were significantly increased compared with control group (P<0.05).The uterine weights,the ratios of uterine weights to body weights,the heights of luminal epithelium,the thickness of myometrium,the amounts of uterine glands and the amount of mature follicle in combination groups with different doses were increased significantly(P<0.05),but had no changes compared with different doses of DES groups (P>0.05). Conclusion: BPA and DES use alone have the estrogenic activities,and the estrogenic effects of co-administered BPA and DES are additive.

Objective:To explore the relationships between ELK-3 and the clinicopathologic features of the patients with hepatocellular carcinoma (HCC) and the effects of ELK-3 on the migration and invasion abilities of human HCC HepG2 cells,and to clarify their mechanisms. Methods:The paraffin-embedded sections of 80 cases of carcinoma tissue and 49 cases of para-carcinoma tissue,fresh postoperative specimens of 18 cases of carcinoma tissue and para-carcinoma tissue were selected.The expressions of ELK-3 protein in HCC and para-carcinoma normal tissues were detected by immunohistochemistry and Western blotting method.The human HCC HepG2 cells at logarithmic growth phase were divided into control-siRNA and ELK-3-siRNA groups.ELK-3 siRNA was transfected into the HepG2 cells,and the migration and invasion abilities of HepG2 were observed by wound-healing assay and Transwell assay. Results: The immunohistochemistry results showed that compared with the para-carcinoma tissue,the positive expression rate of ELK-3 in the HCC tissue was significantly increased (P<0.05),and the positive expression rate of ELK-3 was positively related to the tumor number,TNM stage,Edmondson classification and venous infiltration of the patients (P<0.05).The Western blotting results showed that compared with the para-carcinoma tissue,the expression level of ELK-3 protein in the HCC tissue of 18 HCC patients were markedly increased (P<0.05).The migration distance and the number of invasion cells in ELK-3-siRNA group were remarkably shorter and less than those in control-siRNA group (P<0.05). Conclusion: The expression level of ELK-3 in HCC tissue is significantly increased,and ELK-3 interference could inhibit the migration and invasion of HepG2 cells,which shows that ELK-3 could exert important effects on the occurrence and development of liver cancer by inhibiting the migration and invasion of HCC cells.

Objective:To discuss the influence of Tongfengning Capsule(TFN)in the levels of uric acid(UA),creatinine(Cr),monocyte chemotactic protein-1(MCP-1),tumor necrosis factor-α(TNF-α),xanthine oxidase(XOD) and blood urea nitrogen (BUN) in the serum and the cyclooxygenase-2(COX-2) activity in the kidney homogenate in the gouty nephropathy model mice,and to analyze its improvement effect on the pathological changes of kidney,spleen and thymus tissues in the mice. Methods: The mouse models of gouty nephropathy were set up by using adenine combined with ethambutol.A total of 70 mice were divided into control group,model group,low(200 mg·kg^-1),medium(400 mg·kg^-1)and high(800 mg·kg^-1)doses of TFN groups,allopurinol (50 mg·kg^-1) positive drug group and Tongfengshu Tablets(600 mg·kg^-1) positive drug group (n=10).The levels of serum UA,Cr,MCP-1,TNF-α,XOD,BUN and the COX-2 activity in homoggenate were detected and the histopathological changes of kidney,spleen,and thymus tissues of the mice were observed with HE staining. Results: Compared with control group,the serum levels of UA,Cr,BUN,MCP-1,TNF-α and XOD of the mice in model group were significantly increased (P<0.01),and the COX-2 activity in the homogenate of kidney tissue of the mice was increased obviously (P<0.01).Compared with model group,the serum UA,Cr,BUN,MCP-1,TNF-α and XOD levels of the mice in positive drug groups and high dose of TFN group were significantly decreased (P<0.01),but there was no significant differences in low and medium doses of TFN groups(P>0.05);the activities of COX-2 in homogenate of kidney tissue of the mice in positive drug groups and high doses of TFN group were significantly decreased (P<0.01),but there was no significant differences in the low and medium doses of TFN groups(P>0.05).Compared with control group,the glomerular atrophy of the mice was found in model group,a large amount of urate crystals presented in renal tubules and renal interstitium,the boundaries between thymus cortex and medulla were not clear and the cells were sparse;the demarcation between the red and white pulp of spleen in the mice was not clear,the splenic nodules were reduced in different degrees,and congestion occurred in the splenic sinus.Compared with model group,the pathological changes such as renal glomerular atrophy,spleen sinuse congestion and unclear boundaries between thymus cortex and medulla of the mice in postive drug groups and different doses of TFN groups were improved to some extent. Conclusion: TFN has certain effect in improving the organ lesions and the abnormal biochemical indexes caused by gouty nephropathy in the mice.

Objective:To observe the effects of saponin of litchi seed (SL) on the oxidative stress and nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway in the impaired glucose regulation (IGR) rats,and to clarify their mechanisms. Methods:The IGR rat models were induced by high-fat feeding,then the rats were randomly divided into model group,metformin group (0.20 g·kg^-1),low dose of SL group (0.05 g·kg^<sup>-1),medium dose of SL group (0.1 g·kg^-1),and high dose of SL group (0.20 g·kg^-1); at the same time, normal control group was established; 12 rats in each group. After 6 weeks of administration,the effects of SL on the glucose and lipid metabolism were investigated. The expression levels of superoxide dismutase (SOD),malondialdehyde (MDA),HO-1 mRNA in pancreas tissue of the rats were observed by Real-time PCR,and the expression levels of whole cell HO-1 and Nrf2 in cytoplasm and nucleaus in pancreas tissue of the rats were decected by Western blotting method. The activity of SOD and the level of MDA in pancreas tissue of the rats were detected by the kits. Results: Compared with normal control group,the fasting blood glucose(FBG) and the blood lipid levels of the rats in model group were significantly increased (P<0.05 or P<0.01),the activity and mRNA expression level of SOD in pancreas tissue were decreased (P<0.01),the level and mRNA expression level of MDA were increased (P<0.01),the expression levels of Nrf2 protein in cytoplasm and nucleurs and the nucleur translaocation rate of Nrf2 were increased (P<0.01),and the mRNA and protein expression levels of HO-1 were upregualted (P<0.05 or P<0.01). Compared with model group,the FBG and serum TG levels in metformin group,medium and high doses of SL groups were decreased (P<0.05 or P<0.01),the activities and mRNA expression levels of SOD in pancreas tissue were increased (P<0.05 or P<0.01),the levels and mRNA expression levels of MDA were decreased (P<0.05 or P<0.01),the Nrf2 protein expression levels in cytoplasm were decreased (P<0.05 or P<0.01),the Nrf2 protein expression levels in nucleaus and the Nrf2 nucleaus translocation rates were increased (P<0.05 or P<0.01),and the mRNA and protein expression levels of HO-1 were upregualted (P<0.05 or P<0.01). Conclusion:SL can improve the glycolipid metabolism and has enhancement effect on anti-oxidative stress effect,and their mechanisms might be associated with activating the Nrf2/HO-1 signaling transduction pathway,promoting the nucleurs translocation of Nrf2 and increasing the expression of antioxidant gene in the down-stream such as HO-1.

Objective:To inhibit the expressions of survivin and HIF-1α using RNA interference(RNAi) technique,and to explore their effects on the proliferation and apoptosis of gastric cancer BGC-823 cells. Methods: Two pairs of siRNA were designed against the human survivin and HIF-1α separately.The human gastric cancer BGC-823 cells were transfected with siRNA-survivin,siRNA-HIF-1α and SCR and used as sis group,siH group,and scrambled siRNA (SCR) group;at the same time, blank control group(serum-free medium) was set up.RT-PCR was used to detect the expression levels of survivin and HIF-1α mRNA in the BGC-823 cells in various groups.The BGC-823 cells were transfected with siRNAs using HifectinⅡ.The BGC-823 cells were divided into sis group,sis+siH group,SCR group and blank control group.MTT assay was performed to detect the proliferation activity of cells.The expression levels of survivin and HIF-1α protein in the cells in various groups were detected by Western blotting method.Flow cytometry was used to detect the apoptotic rates of BGC-823 cells in various groups. Results:Compared with blank control group,the expression level of survivin mRNA in sis group was significantly desreased (P<0.05),and the expression level of HIF-1α mRNA in siH group was significantly desreased (P<0.05).Compared with blank control group,the proliferation ability of cells in SCR group had no significant change (P>0.05),and the proliferation abilities of cells in sis group and sis+siH group were significantly decreased(P<0.05);compared with sis group,the proliferation ability of cells in sis+siH group was significantly decreased(P<0.05).Compared with blank control group,the expression levels of survivin and HIF-1α in sis+siH group were significantly decreased (P<0.05 or P<0.01),and the apoptotic rates of BGC-823 cells were significantly increased(P<0.01). Conclusion:The combined targeting silencing surviving and HIF-1α genes can down-regulate the expressions of targeted genes,inhibit the proliferation of gastric cancer BGC-823 cells,and promote the apopotosis,indicating RNAi gene silencing may be a new method to treat gastric cancer.

Objective:To investigate the protective effect of Purendan superfine powder (PRD) on the retinopathy of the diabetic rats and its influence in the nuclear factor kappa B (NF-κB) signaling pathway,and to elucidate its mechanism of action. Methods: Forty Wistar rats were randomly divided into normal control group,model group,low dose of PRD group and high dose of PRD group.After 3 months of administration,the blood sugar levels of the rats in various groups were detected.The expression levels of vascular endothelial growth factor(VEGF) and angiogenin-2(Ang-2) mRNA in retina tissue of the rats were determined by RT-PCR.The expression levels of advanced glycation end products(AGEs),receptor for advanced glycation end products (RAGE),NF-κB,VEGF,Ang-2 proteins in retina tissue of the rats were determined by Western blotting method. Results:Compared with normal control group,the blood sugar level,the expression levels of VEGF,Ang-2 mRNA in retina tissue and the expression levels of AGEs,RAGE,NF-κB,VEGF,Ang-2 proteins in retina of the rats in model group were significantly increased(P<0.05).Compared with model group,the blood sugar levels,the expression levels of VEGF and Ang-2 mRNA in retina tissue,and the expression levels of AGEs,RAGE,NF-κB,VEGF,Ang-2 proteins in retina tissue of the rats in low and high doses of PRD group were significantly decreased(P<0.05). Conclusion: PRD may protect the retina of diabetic rats by specifically blocking the AGEs/RAGE/NF-κB signaling pathway.

Objective:To explore the effects of the different aging time of cured composite resin commonly used in clinic and the silane coupling agent on the bonding strength of metal brackets,and to provide reference for its clinical application. Methods: A total of 20 cuboids were made with denture resin,and 5 resin surfaces were prepared on each cuboid,then 100 specimens of resin for test were obtained.Another 5 cuboids with only one surface were prepared for observation by scanning electron microscope,and the amount of the resin surfaces was 105.After the surfaces were polished with Super-snap discs,the samples were randomly divided into 5 groups; according to different aging time,all of specimens were aged by the method of constant temperature water bath (37℃) in the artificial saliva,and five groups included instant adhesive group (A group),aged 1 h group (B group),aged 1d group (C group),aged 1 week group (D group),aged 1 month group (E group); according to the different surface treatment methods,each bonding group was divided into two groups:coating treatment with silane coupling agent after treated by 33% phosphoric acid group (A1,B1,C1,D1 and E1 groups) and 33% phosphoric acid treatment group (A2,B2,C2,D2 and E2 groups).The specimens were bonded with the metal brackets via 3M chemical cured adhesive,after 4 min solidification,the specimens were put into the artificial saliva and store for 24 h under the temperature of 37℃.The universal mechanical testing machine was used to test the shear bond strengths between the aged composite resins and the brackets,then the adhesive remnant index (ARI) of each test composite resin surface was recorded.Five observation resin surfaces were not treated and the microstructures of different aged composite resin were observed by scanning electron microscope. Results: The shear bond strengths had significant differences between various coating silane coupling agent treatment groups(A1,B1,C1,D1 and E1 groups)(P<0.01); the results of comparison between each two groups showed that the average shear bond strength in A1 group was the highest; the average shear bond strengths in B1 and D1 groups were higher than those in C1 and E1 groups(P<0.01);there were no significantly statistical differences in the average shear bond strengths between other groups(P>0.05);all the shear bond strengths in various groups were higher than the bond strength required in clinic.There were no significantly statistical differences in the shear bond strengths between various phosphoric acid treatment groups(A2,B2,C2,D2 and E2 groups)(P>0.05);only the shear bond strength in A2 group reached the shear bond strength required in clinic;the results of the comparison between each two groups showed that the mean shear bond strength in A2 group was higher than that in C2 group(P<0.05),there were no significantly statistical differences between other groups(P>0.05). There were no statistical differences in ARI of each composite resin surface between silane coupling agent treatment group and phosphoric acid treatment (P>0.05); there were signifieant differences in ARI between groups with the same aging time(P<0.05).The scanning electron microscope results showed that the distribution was nonuniform gradually with the prolongation of time. Conclusion:When the composite resin is instantly bonded with the brackets, the shear bond strength can reach the level in clinic.Sliane coupling agent can increase the bonding strength of aged composite resin to the level required in clinical orthodontic.

Objective:To cover the chitosan(CS) membrane on the biphasic calcium phosphate(BCP) scaffold prepared with 3D printing to prepare the composite scaffold,and to explore its application potentiality in bone tissue engineering. Methods:The BCP scaffold was prepared by 3D printing,and BCP/CS composite scaffold was prepared by covering CS membrane on it.The experiment was divided into control group (BCP scaffold group) and experimental group(BCP/CS scaffold group).The surface roughness and ultrastructures of the scaffolds in two groups were observed by atomic force microscope(AFM) and scanning electron microscope (SEM).The water absorption rates of scaffolds in two groups were measured by mean weighting method.The mouse anterior osteoblasts (MC3T3-E1) were cultured,at the same time,blank group was set up; the toxicity degrees of two kinds of stent extracts after co-cultured with the cells for different time (1,3,and 5d) were determined by CCK-8 method.The proliferation activities of cells at different time (1,3,5,and 7d) were detected by CCK-8 method after the cells were co-cultured with the scaffolds in two groups. Results:The AFM results showed that the surface roughness of the scaffolds in BCP/CS group was lower than that in BCP group.The SEM results showed that the average aperture of the scaffolds in BCP/CS group was 250-300 μm,and the average aperture in BCP group was 350-450 μm.The water absorption rates of scaffolds were(37.75±2.21)% in BCP/CS group and (32.00±2.43)% in BCP group;there was significant difference between two groups(P<0.05).According to the National standard (GB/T16886.5-2003),the toxicities of stent extracts in two groups in 1,3, and 5 d were 0,1, and 1 degree.The MC3T3-E1 cells could adhere and proliferate on the BCP/CS and BCP scaffolds,and the number of the cells in two groups was simply increased with the prolongation of time.The proliferation activity of the cells in BCP/CS group was significantly higher than those in BCP group and blank group (P<0.05) on the 7th day. Conclusion:BCP/CS composite scaffold has the characteristics of bone tissue engineering scaffold material without cytotoxicity and can improve the cell proliferation ability.It has the potential as scaffold of bone tissue engineering.

Objective:To investigate the effect of polycystin-1(PC1)on the apoptosis of human cervical cancer HeLa cells,and to provide the basis for further clarifying the effect of PC1 on apoptosis. Methods: The recombinant plasmid pEGPF-PC1-5TMC of PC1 gene and blank control plasmid pEGFP were respectively used to transfect the HeLa cells,which was verified by Western blotting method.The proliferation activities of cells in transfection group (transfected with recombinant PC1 plasmid),blank control group(transfected with blank control plasmid pEGFP) and normal control group(normal HeLa cells)were respectively detected by MTT at 48 and 72 h after transfection.The apoptotic rates of the cells in transfection group and blank control group were detected by TUNEL method. Results: Compared with normal control group,the proliferation ability of HeLa cells in transfection group had no significant difference at 48 and 72 h after transfection(P>0.05);compared with blank control group,the proliferation ability of HeLa cells in transfection group was increased significantly (P<0.05) at 48 and 72 h after transfection.The apoptotic rate of HeLa cells in transfection group was obviously lower than that in blank control group (P<0.01). Conclusion: PC1 gene has anti-apoptosis effect on the human cervical cancer HeLa cells.

Objective:To evaluate the clinical value of heart rate variability (HRV) in evaluation on the short-term major adverse cardiac events (MACE) in the patients with acute myocardial infarction (AMI) after percutaneous coronary intervention (PCI). Methods:A total of 160 AMI patients received PCI were collected and divided into control group (126 cases,there was no MACE during 3 months after PCI) and research group (34 cases,there was MACE during 3 months after PCI) according to the occurrence of MACE after operation. The main HRV indexes including SDNN,SDANN,SDNNIDX,PNN50,RMSSD,LF and HF of the patients in two groups were compared. The Gensini scores of coronary artery of each patient were calculated. The relationships of the main HRV indexes and the Gensini scores of coronary artery were analyzed. After 3 months of follow-up,the risk factors of the main HRV indexes to the occurrence of MACE were confirmed by multivariate Logistic analysis. Results: The main HRV indexes including SDNN,SDANN,SDNNIDX,PNN50,RMSSD and HF of the patients in research group were lower than those in control group (all P<0.05),but LF in research group was higher than that in control group (P<0.05).The negative relationships between SDNN,SDANN,SDNNIDX and the Gensini scores were confirmed (r=-0.827,r=-0.789,r=-0.698, P<0.05),but rMSSD,pNN50,LF and HF had no relationship with the Gensini scores (P>0.05). The multivariant Logistic analysis showed that SDNN,SDANN,SDNNIDX were the independent protective factors to the occurrence of MACE (P<0.05). Conclusion:The HRV indexes indicating the sympathetic nervous system function can inflect the degree of coronary artery stenosis and assess the short-term prognosis in the AMI patients after PCI.

Objective:To explore the relationships between the levels of autophagy-related protein 9a (ATG9a) in the plasma and the end-diastolic interventricular septum dimension(IVSd) and left ventricular posterior wall dimension (LVPWd) in the hypertension patients with left ventricular hypertrophy (LVH),and to further evaluate its effect in clinical prediction for the hypertension patients with LVH. Methods: One hundred and twenty-eight hypertension patients in hospital were enrolled.According to the results of cardiac echocardiography,all enrolled patients were divided into LVH group (n=66) and non-LVH group (n=62).The levels of plasma ATG9a in the patients ware detected by enzyme linked immunosorbent assay (ELISA).The levels of plasma ATG9a of the patients in LVH group and non-LVH group were compared.The correlations between the levels of plasma ATG9a and IVSd or LVPWd in the hypertension patients with LVH were further analyzed.And the area under receiver operating characteristic curve (AUC) of plasma ATG9a level was calculated. Results:The plasma ATG9a level of the hypertension patients in LVH group was higher than that in non-LVH group (P<0.05).The plasma ATG9a level in the patients with LVH showed significantly positive correlations with IVSd and LVPWd(r=0.774,r=0.685,P<0.01). The AUC for plasma ATG9a level was 0.976(95%CI:0.923-0.996).The optimal cut-off value of plasma ATG9a level for ROC curve was 1.69 μg·L^-1,the sensitivity and specificity were 96.8% and 90.6%,respectively. Conclusion:The plasma ATG9a may play an important role in the pathogenesis of myocardial hypertrophy as autophagy-induced factor. The level of plasma ATG9a has positive correlation with IVSd and LVPWd in the hypertension patients with LVH. The level of plasma ATG9a may predict the LVH in the hypertension patients.

Objective:To detect the expression levels of angiogenin-1, 2 (Ang-1, 2), vascular endothelial growth factor (VEGF), tumour necrosis factor (TNF-α) and interleukin-6 (IL-6) in the serum and villus tissue of the patients with missed abortion before and after taking mifepristone,and to elucidate the mechanism of mifepristone for the treatment of missed abortion. Methods: A total of 68 cases of patients with missed abortion were selected as drug abortion group,and 51 cases of healthy pregnant women were selected as control group. ELISA was applied to detect the levels of Ang-1, Ang-2, VEGF, TNF-α, and IL-6 in serum of the patients; immunohistochemistry was used to detect the expression levels of Ang-1, Ang-2, VEGF, TNF-α, and IL-6 in villus tissue. Results:The levels of Ang-1, Ang-2, VEGF, TNF-α and IL-6 in serum of the patients in drug abortion group after abortion were higher than those before abortion(P<0.05).The expression levels of Ang-1, Ang-2, VEGF, and TNF-α in drug abortion group after abortion were higher than those in control group(P<0.05), but the IL-6 expression level was lower than that in control group (P<0.05). The levels of Ang-1 and Ang-2 in serum and villus tissue had positive correlation with the VEGF level(r=0.849,r=0.721,r=0.590,r=0.532,P<0.05). Conclusion:Mifepristone can promote the termination of missed abortion by increasing the levels of Ang-1, Ang-2, VEGF, and TNF-α in serum and villus tissue and decreasing the IL-6 level.

Objective:To investigate the expression of tumor protein P53(TP53) in the villus tissue of the patients with unexplained recurrent spontaneous abortion(URSA) and the effects on the apoptosis and migration of human trophoblast cells,and to clamify their mechanisms preliminarily. Methods:The villus tissues of 40 patients with URSA(patient group) and 30 healthy pregnant women underwent artificial abortion(control group) were selected.The expression levels of TP53 mRNA and protein in the villus tissue were detected by RT-PCR and Western blotting method.The human trophoblast HTR-8 cells were divided into blank control group,vector group and TP53-siRNA group.Western blotting method was employed to detect the expression levels of TP53 protein in cells in three groups after transfected with siRNA.The apoptotic rates, the number of migrated cells and the expression levels of TP53,Cleaved-Caspase3,Bcl-2, and Bax proteins in blank control group and TP53-siRNA group were detected respectively by flow cytometry,Transwell assay and Western blotting method. Results:The mRNA and protein expression levels of TP53 in the villus tissue of the patients in patient group were significantly higher than those in control group (P<0.01). The TP53 protein expression level in cells in TP53-siRNA group was significantly lower than that in blank control group(P<0.01).After transfection for 48 h, the apoptotic rate in TP53-siRNA group was significantly lower than that in blank control group,the number of migrated cells was significantly higher than that in blank control group (P<0.01),the expression levels of Cleaved-Caspase3 and Bax proteins in TP53-siRNA group were significantly lower than that in blank control group,and the expression level of Bcl-2 protein was significantly higher than that in blank control group (P<0.01). Conclusion: TP53 highly expresses in the villus tissue of the patients with URSA;silencing of TP53 expression in HTR-8 cells can significantly promote the migration of human trophoblast cells and inhibit apoptosis.

Objective:To understand the distribution of Rh blood group system E antigen (RhE) in the patients before blood transfusion in clinic,and to analyze the clinical significance of detection of RhE antigen combined with the detection frequencies and the features of anti-E antibodiy in the patients. Methods:Microcolumn gel method was used to detect the RhE antigen in 10 000 cases of hospitalized patients before blood transfuion,and irregular antibody screening and identification of the samples were performed. Results: The positive rate of RhE antigen in 10 000 cases of samples was 51.46% (5 146/10 000),the negative rate was 48.54% (4 854/10 000).There were 78 cases of positively irregular antibody screening in the patients,and the positive rate was 0.78%(78/10 000).There were 18 cases of anti-E antibody,accounting for 23.08% (18/78).In the 18 cases of patients with positive anti-E antibody,there were 17 cases of patietns with history of blood transfusion and pregnancy. The detection rate of anti-E antibody in the patients with history of clinical immunization was 0.33%(17/5 120,which was significantly higher than that in the patients without history of immunization (0.029%,1/4 880)(χ²=13.46,P<0.01). Conclusion: Anti-E antibody is most common irregular antibody in clinical blood transfusion detection,The detection of RhE antigen in the patients before transfusion can realize the RhE same type matching blood transfusion,which can avoid to produce the anti-E antibody in the patients and promote the clinical transfusion safety.

Objective:To discuss the clinical manifestations and early combined diagnosis of subacute combined degeneration(SCD)of the spinal cord. Methods:The clinical data of 18 patients with SCD were collected,and the clinical manifestations and the levels of serum vitamin B12(VB12)and homocysteine(Hcy),mean corpuscular hemoglobin(MCH) levels,and mean corpuscular volume (MCV) were detected.The abnormal appearence of electrophysiological examination of the SCD patients was analyzed. Results:There were 12 cases of limb numbness in 18 SCD patients,10 cases of motor disturbance,1 case of blurred vision,4 cases of mental abnormality,and 2 cases of urinary disturbance.Among the 18 patients,the serum VB12 levels were decreased in 9 cases (50.0%),the Hcy levels were increased in 18 cases (100%),the MCV were increased in 15 cases(83.3%) and the MCH levels were increased in 14 cases (77.8%).The results of neuroelectrophysiological examination in 15 patients were all positive. Conclusion:The patients with SCD lack of the characteristic clinical manifestations.The diagnosis rate of SCD is low by the lack of serum VB12,and the combined detection of serum VB12,Hcy,MCV,MCH and electrophysiological examination is of great significance for the early diagnosis of SCD.

Objective:To compare the clinical efficacy and safety of ticagrelor and clopidogrel that applied in the antiplatelet therapy in the patients after coronary artery bypass grafting(CABG),and to provide a more rational strategy for antiplatelet therapy. Methods:A total of 260 patients underwent CABG were selected randomly and divided into ticagrelor group (n=129) and clopidogrel group (n=131). The patients in ticagrelor group were given ticagrelor 90 mg·d^-1 and aspirin 100 mg·d^-1,and the patients in clopidogrel group were given clopidogrel 75 mg·d^-1 and aspirin 100 mg·d^-1.After 12 months of follow-up,the major adverse cardiovascular events (MACE) and the adverse events related to safety were collected such as cardiovascular death,non-cardiovascular death,recurrent myocardial infarction,re-admission to hospital due to recurrent unstable angina pectoris and heart failure,dyspnea,bleeding,etc.The data was analyzed by statistical method. Results:In this study,241 patients finished the follow-up plan,including 122 patients in ticagrelor group and 119 patients in clopidogrel group.In the aspect of MACE,there were 10 cases (8.2%) in ticagrelor group and 19 cases (16.0%) in clopidogrel group; the difference between two groups was not statistically significant (P>0.05). In ticagrelor group,one patient (0.8%) came back to hospital again because of heart failure,and six cases (5.0%) in clopidogrel group; the difference between two groups was statistically significant (P<0.05). In the safety related adverse events aspect,there were 27 cases (22.1%) in ticagrelor group and 17 cases (14.3%) in clopidogrel group; the difference between two groups was not statistically significant (P>0.05). Conclusion: The efficacy and safety of ticagrelor applicated in the patients after CABG are not different compared with clopidogrel,which can be used as the antiplatelet strategy after CABG.

Objective:To analyze the curative effects of three inlays with different materials in repairing the poserior teeth class Ⅱ cavity by clinical indexes, and gigival indexes, and to provide the basis for choosing the proper repairing method for poserior teeth class Ⅱ cavity. Methods:According to patients' appeal and willing,120 poserior teeth class Ⅱ cavity from 120 patients were divided into three groups(40 teeth in each group),and were restored by metal, ceramic and resin inlays.The contralateral teeth of ill teeth or the corresponding jaw teeth with the same name were used as control group.The clinical indexes were observed one year later,and the gigival index(GI) and the plaque index(PI) were calculated.The curative effects of three inday restorations were compared according the clinical evaluation criteria one year later. Results: Compared with control group,the GI and PI of the patients in metal inday group had significant differeces(P<0.05),and were higher than those in ceramic inlay group and resin inlay group(P<0.05).Compared with control group,the GI and PI of the patients in ceramic inlay group and resin inlay group had no significant differences(P>0.05).The clinical curative effects of ceramic and resin inlays were better than that of metal inlay after one year restoration of posterior teeth class Ⅱ cavity. Conclusion: Three materials of inlays have some stimulation on the gigiva with different extents in repairing the poserior teeth cavity class Ⅱ cavity. The metal inlay has most stimulation, resin inlay is the next, and ceramic inlay has least stimulation. Ceramic and resin inlays have better reparing effects than metal inlay.

Objective:To explore the clinical characteristics of the Kawasaki disease children with joint involvement,and to determine whether joint involvement will affect the coronary artery damage rate, intravenous immunoglobulin (IVIG) reactivity and prognosis in the children with Kawasaki disease. Methods:A total of 636 cases of children with Kawasaki disease with clear diagnosis and complete informations were selected.According to whether there were the local symptoms of the joints or not,they were divided into joint involvement group(n=31) and non-joint involvement group(n=605).The clinical and auxiliary examination data of the Kawasaki disease children were retrospectively analyzed.The clinical features,laboratory indexes,coronary artery damage rate and IVIG response rate of the children were compared between joint involvement group and non-joint involvement group. Results:Among 31 Kawasaki disease child patients with joint involvement,there were 25 cases(80.6%)of children more than 3 years old,26 cases(83.9%)) diagnosed with complete Kawasaki disease,24 cases(77.4%) of multiple joint involvement (≥ 2 joints),26 cases(83.9%) of big joint involvement,and the knee joint involvement was more common (20 cases,64.5%).The duration of joint local symptoms varied,and no local joint sequelae occurred after follow-up.The fever time of the children in joint involvement group was longer than that in non-joint involvement group,and the difference was statistically significant(P<0.05).The number of WBC,neutrophil percentage and C-reactive protein level of the children in joint involvement group were higher than those in non-joint involvement group,and the difference was statistically significant(P<0.05).There was no significant difference in coronary vascular damage rate between two groups(P>0.05).The IVIG non-response rate of the children in joint involvement group was higher than that in non-joint involvement group(39.3% vs 10.9%),and the difference was statistically significant(P<0.05). Conclusion:Kawasaki disease with joint involvement mostly occurs in the children over 3 years old.When Kawasaki disease accompanies with joint involvement,it tends to multiple joint involvement(≥ 2 joints),more common in the big joint,especially in knee joint involvement. Kawasaki disease accompanying with joint involvement has no affect on the incidence of coronary artery lesions,but it behaves poor reactivity to IVIG.Joint involvement is a transient symptom of Kawasaki disease,and there are no sequelae occurred.

Objective:To study the clinical features of skin metastatic carcinoma,and to provide the reference for its diagnosis and treatment. Methods:The clinical materials of one case of cutaneous metastases complicated with herpes zoster were analyzed and the related literatures were reviewed.The right chest wall in the patient appeared erythema and blisters with pain 4 months ago;in the local hospital the patient was diagnosed as herpes zoster and received relevant treatment;the pain was improved,but there were still erythema,papules and nodules in the local area.The skin lesions were erosive,expanded and deepened rapidly 20 days ago,and the pathological examination,lung CT,abdominal CT and other assistant examinations were performed in our hospital. Results:The lesion biopsy results showed invasive carcinoma with extensive infarction.The results of lung CT,abdominal CT and other assistant examinations showed the right breast occupying lesions complicated with cervical lymph nodes,liver,pleural and bone metastases.The skin lesions were improved after local treatment and 2 courses of chemotherapy(docetaxel combined with trastuzumab), but failed to heal completely. Conclusion: Skin metastatic carcinoma is rare,and its clinical manifestations are different.The clinicians should master the regularity of the disease,timely conduct the histopathological examination and the related examination in the other parts to reduce the incidence of misdiagnosis and missed diagnosis.

Objective:To use 3D printing titanium alloy pelvic prosthesis to reconstruct the pelvis in a patient with metastatic acetabulum tumor, and to explore a novel method in the treatment of acetabulum tumor. Methods: One patient with metastatic acetabulum tumor was selected.The pelvic CT scan was conducted in the patient.Three-dimensional reconstruction of pelvic CT was conducted using Mimics 14.0 Software. The 3D printing stereo lithography apparatus resin pelvic model with 1:1 scale was manufactured. The 3D printing osteotomy guide was designed and manufactured in accordance with the preoperative designed osteotomy scope; 3D printing titanium alloy pelvic prosthesis was designed on the basis of mirror image normal side hemi-pelvis and manufactured by electron beam melting 3D printing technology. After the osteotomy assisted with 3D printing osteotomy guide,pelvic prosthesis was implanted to the designed location. Results: Several follow-ups were reviewed in the patient within postoperative 12 months. The results of X-ray image showed there were no signs of pelvic prosthesis displacement and obvious migration.The SF-36 score(the MOS item shorted from health survey)was 75.35 points at 12 months after operation; MSTS was 11 points at 12 months after operation.The Harris score of affected side hip joint was 52 points at 12 months after operation.The motion function of lower limbs after operation of the patient was restored.The daily actions such as walking and standing could be done by herself with the assistance of crutch. Conclusion: In this study,3D printing pelvic prosthesis achieve the accurate reconstruction of pelvis and ameliorate the postoperative motion function of the patient.

Objective:To establish a drug concentration in plasma determination method based on fluorescence polarization immunoassay (FPIA),and to explore the feasibility of this method in monitoring the concentrations of three small molecular drugs including gentamycin,digoxin and sodium valproate in plasma of the mice. Methods:The standard samples of gentamycin,digoxin,and sodium valproate were selected. The limits and linear ranges of drugs were measured with fluorescence polarization(FP) non-linear fitting method. Three standard groups with low,medium,and high concentrations of drug were selected during liver range,and the accuracy,inter-day precisions and serum anti-interference abilities of the mice were detected.Total 30 KM mice were divided into gentamycin group (5 μg·g^-1,intramuscular injection),digoxin group(20 ng·g^-1,gavage) and sodium valproate group(15 μg·g^-1,gavage); the blood drug concentrations of the mice were measured at 1,3,6,12 and 24 h time points after administration. Results:For all three drugs detected,the detection limits detected by FPIA were lower than 50 μg·L^-1),the linear ranges were broad (more than 10 times of detection limit),the accuracy (ER) was lower than <5% and the inter-day precisions were higher (CV<5% in medium and high concentration groups).The mouse serum showed no affection to FPIA detection qualities for gentamycin and digoxin,while high concentration of serum negatively affected the detection quality for sodium valproate (ER=-12.84% in 50% serum group).After administration,gentamycin reached the peak blood concentration at 1 h with half-life period of 3 h,digoxin reached the peak blood concentration at 1 h with half-life period of >24 h,and sodium valproate reached the peak blood concentration at 3 h with half-life period of 6-12 h. Conclusion: FPIA for small molecular drug concentration in plasma of the mice has the advantages of small sample size,high accuracy,simple preprocessing,high throughput detection and rapid test,which can be applied in determination in pharmacological,pharmaceutical and clinical research.

Objective:To analyze the gene specificities of mitochondrial cytochrome b (Cytb) of velvet antler and cytochrome C oxidase subunit Ⅰ (COⅠ),and to establish the duplex polymerase chain reaction(PCR)technique for identifying the molecular fingerprint characteristics of velvet antler. Methods:A modified alkaline method was used to extract the genomic DNA from the pilose antler of Cervus Nippon Temminck,Cervus elaphus Linnaeus,Rangifer tarandus and New Zealand deer.Based on Cyt b and COⅠ genes,the specific primers were designed with Premier 5.0 software(Cytb1,2 and CO Ⅰ 1,2,3);PCR amplification was carried out by the single and duplex primers,and the best PCR conditions and highly specific primers were determined. Results:The length of genomic DNA fragment extracted by the modified alkaline method was 23 000bp,and the DNA purity was 1.80±0.02;PCR amplification by the single primer did not identify the authenticity of velvet antler;when Cytb1 and COⅠ1 were applied to PCR, the annealing temperature was 58℃,the fragments of 395 bp and 525 bp were specifically amplified from both Cervus Nippon Temminck and Cervus elaphus Linnaeus antler(Jilin,Anhui),but no fragment appeared for the Rangifer tarandus and New Zealand pilose antler. By using the determined extraction method and the optimal PCR condition,the commercial velvet antlers were carried out and the test results were identical with the reality. Conclusion:The duplex PCR technique can distinguish the authenticity of velvet antler from the molecular level.The method is good in specificity,practicability,and simplicity,and has high application value in the identification of velvet antler.

Objective:To establish the culture method of the human amnion fibroblasts in vitro and to identify based on the cell morphology and immunohistochemistry to get the human amnion fibroblasts,and to lay the foundation for the study on stem cells in the future. Methods:The amnion membrane was removed from the placenta of the parturia,after digestion of trypsin and collagenase,the human amnion fibroblasts were isolated,and DMEM culture including epidermal growth factor(EGF) was used to culture the cells.The morphology of cells was observed by microscope.HE staining and immunohistochemistry were used to analyze the cell characteristics.The purity of human amnion fibroblasts was analyzed by flow cytometry(FCM). Results:After digested with trypsin and collagenase, the human amnion fibroblasts were obtained,and the cells were found in a radial or vortex-like form under microscope.The immunohistochemistry results showed that the human amnion fibroblasts obviously expressed the fibroblast markers Vimentin,expressed the ion binding protein A4 (S100A4) in different degrees,and did not express the epithelial marker keratin CK19.The FCM results showed that the purity of human amnion fibroblasts was 86.1%. Conclusion:The isolation and identification method of human amnion fibroblasts is established successfully,and they have the specific markers and phenotypic characteristics of fibroblasts.

Objective:To establish a simple and efficient method for primary culture and identification of the rat colon fibroblasts in vitro,and to provide cell model for the further study on colon fibrotic diseases. Methods:The colon tissue of adult male Wistar rats was selected. The rat colon fibroblasts were cultured by tissue explant method,and isolated by trypsinzation combined with different speed attaching.The morphology of fibroblasts was observed by HE staining.Immunohistochemistry staining was used to observe the expressions of Vimentin,α-smooth muscle actin (α-SMA),S100 calcium-binding protein A4(S100A4) and E-cadherin in the cells.The fibroblasts were identified by comparison with the smooth muscle A7R5 cells of thoracic aorta of the rats. Results:The fibroblasts began to climb from the tissue at the 8th day,they began to proliferate at the 12th day of the culture,and grew throughout the culture flasks at the days 15-20.The fibroblasts were flat polygonal spindle or starlike under inverted microscope.The HE staining results showed that the nuclei of fibroblasts were large,oval and pale colored and had prominent nucleoli.The results of immunohistochemistry showed that the expression of Vimentin in fibroblasts was positive,and the expressions of α-SMA,S100A4 and E-cadherin were negative.The expression of S100A4 in the smooth muscle A7R5 cells of thoracic aorta of the rats in control group was negative,and the expressions of α-SMA,Vimentin and E-cadherin were positive. Conclusion:The rat colon fibroblasts can be successfully cultured by trypsin combined with explant culture method.