BACKGROUND:In vitro culture of sufficient vaginal epithelial cels and smooth muscle cels is the key for vaginal tissue engineering. However, the culture, purification and passage of vaginal epithelial celsin vitro are difficult. Primary culture and passage of vaginal epithelial cels from large animals such as canines has not been reported. OBJECTIVE:To establish a stable method of culturing canine vaginal epithelial cels and smooth muscle cels. METHODS: Vaginal epithelial cels were isolated from the vaginal specimens by enzymatic digestion with Dispase and trypsin separately, and cultured in keratinocyte serum-free medium. Vaginal smooth muscle tissue were minced and digested with colagenase type II; the colected smooth muscle cels were cultured in DMEM culture medium containing 10% fetal bovine serum. The cultured cels were passaged regularly. Cel morphology and proliferation characteristics were observed and cel phenotypes were confirmed by morphology and immunohistochemistry staining. RESULTS AND CONCLUSION: Primary vaginal epithelial cels began to adhere after 24-36 hours, grew logarithmicaly after 4-5 days, and reached 70% confluence after 7-8 days; the epithelial cels showed a typical cobblestone, with no fibroblasts. Cultured epithelial cels passaged every 4-5 days and subcultured to 6-7 generations continuously. Immunohistochemical staining confirmed a positive staining for anti-pancytokeratin (AEl/AE3). Primary cultured smooth muscle cels adhered and grew after 24 hours. The smooth muscle cels were spindle-shaped and proliferated logarithmicaly. After 4 days, primary cultured smooth muscle cels were confluent and showed a typical shape of “peaks and valeys”, and then the cels could be passaged every 3-4 days and passaged 7-8 generations. Immunohistochemistry staining showed α-actin staining was positive. These findings indicate that canine vaginal epithelial cels and smooth muscle cels could have a long-term stable culture and proliferation, to provide adequate seed cels for vaginal tissue engineering.

Purpose To study NF-κB gene expression level in mouse hepatocellular carcinoma cell lines with differently lymphatic metastasis potentials and to discuss its roles in lymphatic metastasis.Methods Using real-time quantitative PCR, NF-κB gene expression level was detected in mouse hepatocellular carcinoma cell lines, including Hca-P with low lymphatic metastasis potential and Hca-F with high lymphatic metastasis potential.Results NF-κB mRNA expression in Hca-P and Hca-F cell lines were (1.41±0.48)×10~(-3),and (2.95±0.22)×10~(-3) (P<0.01),respectively.NF-κB mRNA expression levels were increased with metastasis potential.Conclusion NF-κB gene may play an important role in lymphatic metastasis of hepatocellular carcinoma.

Objective To evaluate the feasibility of bladder extracellular matrix (BACM) seeding with autologous cultured vaginal smooth muscle cells (VSMC) repaired rabbit vaginal defect.Methods This study included 24 female rabbits.BACM and vaginal acellular matrix (VACM) were obtained from 8 rabbits by decellularization process.The other 16 rabbits were randomly divided into experimental and control groups.Vaginal tissue biopsies ( ～ 1 cm2) were harvested from female New Zealand rabbits.VSMC were cultured and stained with a-smooth muscle actin antibodies. Cultured VSMC cells were seeded on BACM ( experimental group) or VACM ( control group) at a cell density of 1 × 107 cells/cm2.The cell-seeded matrixes were cultured for 5days.In experimental group of 8 rabbits,a 2 cm segment of vagina was resected and replaced with BACM seeding with VSMC.Then the regenerative segment was studied with histological technique by hematoxylin-eosin staining after 3,6 and 12 weeks postoperative aud vaginography was performed at 12 weeks postoperative.The 8 rabbits in control group underwent the exact same procedure as above but the vaginal defect was repaired with VACM seeding with VSMC,Results The prepared BACM and VACM were transparent,HE staining and scanning electron microscopy showed the two acellular matrix was both consisted of abundant network of fibers with the regular arrangement,without cellular debris.Primary culture of VSMC was successfully established and passaged,and was uniformly spindle-shaped in the confluent state and showed a characteristic hill and valley'formation.Immunohistochemical staining showed VSMC in culture stained positively with a-smooth muscle actin antibodies.VSMC began to adhere to the BACM 5 hour after implanting and the number of the adhered cells increased with time.Cells gradually expanded and showed the typical morphology of smooth muscle cells.Three weeks after implantation in vivo,the luminal surface of matrix was completely covered by vaginal epithelial tissue,a layer of smooth muscle cells was formed in the outer surface of the matrix.Multilayered vaginal epithelial and improved development of organized muscle bundles was observed after 6 weeks.The regenerative tissue was equivalent to the normal vaginal tissue at 12 weeks postoperatively.Vaginography demonstrated the maintenance of full patency and a wide vaginal caliber without fibrosis and graft rejection.There was no significant difference in all evaluated items between experimental and control groups.Conclusions BACM has the same regenerative process as VACM in the replacement of vaginal defect.Moreover,BACM has wider source and appears to be an suitable material for vaginal replacement.

BACKGROUND:Human has a high level heritability in physical performance. With the development of technology and test method in molecular biology, the researchers of sport science are concerned with the influence of gene variation on the elite athlete performance. They begin to know the important value of gene on predicting the physical performance.
OBJECTIVE:To review the research results in the field of gene polymorphisms and elite athlete performance and to expatiate the problems in these researches, thereby offering some proposals.
METHODS:A computer-based online research of PubMed and CNKI databases was performed to col ect articles published from 1998 to 2013 with the key words“elite athlete performance, gene polymorphisms, endurance, power, training response”in Chinese and English. There were 150 articles after the initial survey. A total of 80 articles were included according inclusion and exclusion criteria.
RESULTS AND CONCLUSION:The researches of this field are mainly focused on the three aspects:elite endurance performance, elite power performance, and training response, which are associated with gene polymorphisms. The main genes related to elite endurance performance are ACE, mtDNA, PPAR, ADR, GNB3, NRF2, etc. The main genes related to elite power performance are ACTN3, ACE, GDF-8, IL-6, HIF-1, etc. The main genes related to training response are HBB, TFAM, NRF2, AR, FECH, etc. Several gaps in the current researches have been identified including smal sample size of most athletic cohorts, lack of corroboration with replication cohorts of different ethnic backgrounds. The numerous research findings can be applied to the gene selection of athletes by creating some kinds of algorithms and models.

Objective The purpose of this study was to investigate the mechanism of action of polypeptide extracts of Eupolyphaga sinensis Walker ( ESW) against oxidative aging.Methods Mice were intraperitoneally injected D-galac-tose for consecutive 20 days to establish an aging mouse model.The model mice were administered with different doses of ESW polypeptide (0, 40, 80, 160 mg/kg/d).The normal activity, movement and anti-stress ability of the mice were ob-served.The activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX) in blood and different tissues and the content of glutathione ( GSH) and malondialdehyde ( MDA) of the aging mice were assessed by xanthin oxidase activity measurement and spectrophotometry, respectively.The expression of nuclear factor erythroid 2-re-lated factor 2 (Nrf2) in Caco-2 cells was detected by immunofluorescence.Results Comparing the control and polypep-tide groups, there were significant decreases of body weight gain, organ indexes, anti-stress ability and activity capacity, the activity of SOD, CAT, GSH-PX and the content of GSH, and an increase of the content of MDA in blood and different tissues in the aging mice.With the increasing dose of polypeptide extracts of ESW, the body weight gain, organ indexes of the liver, spleen and kidney were significantly increased, the static and dynamic exercise time was prolonged in the poly-peptide group, and their abilities of hypoxia tolerance and heat tolerance were close to that of normal controls.The SOD, CAT, GSH-PX activity and GSH level in blood and different tissues were significantly increased, but MDA content de-creased.The expression of Nrf2 in Caco-2 cell nuclei was significantly increased in the polypeptide group, close to that of the positive control group.Conclusions The results of our study show that polypeptide extracts of ESW improve the anti-stress and antioxidative capacity in D-galactose-induced mouse models of oxidative aging by initiating Nrf2-ARE antioxidant signaling pathway, therefore, delay the oxidative aging in mice.

Objective To determine the relationship of cytochrome P-450 epoxygenase metabolites of arachidonic acids with impaired endothelium-dependent vasorelaxation in the healthy elderly. Methods A case-control study was employed.The study enrolled 60 healthy young adults (group A) and 60 healthy senior citizens (group B) of Han population in Shanghai. Serum contents of 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) (a stable metabolite of 14,15-EET), 6-Kote-PGF1a (a stable metabolite of prostaglandin) and TXB2 (a stable metabolite of thromboxane A2) were measured using ELISA kits. The endothelium-dependent vasorelaxation (EDV) and endothelium-independent vasorelaxation (NEDV) in brachial arteries were determined by color Doppler ultrasound. Results Compared with group A, group B had significant higher levels of hemoglobin A1c, triglyceride and cholesterol levels (P＜0.01), and significant lower levels of 14,15-DHET and 6-Kote-PGF1a (P＜0.001), leading to increased values of TXB2/6-Kote-PGF1a and TXB2/14,15-DHET. There was bigger basal interior diameter of brachial arterials with reduced EDV and NEDV response (P＜0.01 and P＜0.05, vs. group A respectively) in group B. Moreover, the age was negatively correlated with 14,15-DHET and TXB2/14,15-DHET. Conclusions Our results indicate that the impaired EDV and NEDV in aging are associated with reduced production of arachidonic acid metabolites through cytochrome P-450 epoxygenase pathway and cyooxygenase pathway of endothelium in the healthy elderly.

Objective To assess the mortality and risk factors for stroke among dialysis patients with different dialysis modality. Methods 590 patients who underwent hemodialysis (HD) or peritoneal dialysis (PD) from January 2008 to December 2012 were recruited in our study, and categorized according to dialysis modality. The prognostic risks of stroke were hazard ratio of risk was calculated by Cox regression analysis in HD and PD patients respectively. by the Kaplan?Meier curves or the Cox proportional hazards model. Results A total of 590 patients is under a median follow?up of 32.5 months. The stroke incidence rate of 49.2/1, 000 patient?years in total patients, and 74.1/1, 000 patient?years in HD patients, which was significantly higher compared with that of 31.8/1,000 patient?years in PD patients. On multivariate analysis, independent predictors of stroke occurrence were age(HR=1.05;95%CI:1.02~1.09;P=0.003)、diabete(HR=1.98;95%CI:1.31~3.46;P=0.001)、CVD(HR=2.06;95%CI:1.62-3.05;P < 0.001)、Total triglycerides(HR = 1.20; 95% CI:1.08-1.58; P = 0.034) and hemodialysis (HR = 2.03; 95% CI:1.46-3.89; P = 0.005). Conclusions Age, diabete, CVD, total triglycerides and hemodialysis are independently associated with increased stroke risks in dialysis patients, which suggest that these patients should pay attention to weight control and glucose control.

Objective:Trigeminal neuralgia(TN)is a severe chronic neuropathic pain that mainly affects the distribution area of the trigeminal nerve with limited treating efficacy.There are numerous treatments for TN,but currently the main clinical approach is to suppress pain by carbamazepine(CBZ).Brain-derived neurotrophic factor(BDNF)is closely related to chronic pain.This study aims to determine the effects of CBZ treatment on BDNF expression in both the trigeminal ganglion(TG)and serum of TN via a chronic constriction injury of the infraorbital nerve(ION-CCI)rat model. Methods:The ION-CCI models were established in male Sprague-Dawley rats and were randomly divided into a sham group,a TN group,a TN+low-dose CBZ treatment group(TN+20 mg/kg CBZ group),a TN+medium-dose CBZ treatment group(TN+40 mg/kg CBZ group),and a TN+high-dose CBZ treatment group(TN+80 mg/kg CBZ group).The mechanical pain threshold in each group of rats was measured regularly before and after surgery.The expressions of BDNF and tyrosine kinase receptor B(TrkB)mRNA in TGs of rats in different groups were determined by real-time PCR,and the expression of BDNF protein on neurons in TGs was observed by immunofluorescence.Western Blotting was used to detect the protein expression of BDNF,TrkB,extracellular regulated protein kinases(ERK),and phospho-extracellular regulated protein kinases(p-ERK)in TGs of rats in different groups.The expression of BDNF in the serum of rats in different groups was detected by enzyme-linked immunosorbent assay(ELISA). Results:The results of mechanical pain sensitivity showed that there was no significant difference in the mechanical pain threshold in the right facial sensory area of the experimental rats in each group before surgery(all P>0.05).From the 3rd day after operation,the mechanical pain threshold of rats in the TN group was significantly lower than that in the sham group(all P<0.01),and the mechanical pain threshold of rats in the TN+80 mg/kg CBZ group,the TN+40 mg/kg CBZ group,and the TN+20 CBZ mg/kg group was higher than that in the TN group(all P<0.05).The BDNF and TrkB mRNA and protein expressions in TGs of rats in the TN group were higher than those in the sham group(all P<0.05),and those in the TN+80 mg/kg CBZ group,the TN+40 mg/kg CBZ group,and the TN+20 mg/kg CBZ group were lower than the TN group(all P<0.05).The p-ERK levels in TG of rats in the TN+80 mg/kg CBZ group,the TN+40 mg/kg CBZ group,and the TN+20 mg/kg CBZ group were significantly decreased compared with the TN group(all P<0.05).The BDNF and neuron-specific nuclear protein(NeuN)were mainly co-expressed in neuron of TGs in the TN group and they were significantly higher than those in the sham group(all P<0.05).The co-labeled expressions of BDNF and NeuN in TGs of the TN+ 80 mg/kg CBZ group,the TN+40 mg/kg CBZ group,and the TN+20 mg/kg CBZ group were lower than those in the TN group(all P<0.05).The results of ELISA showed that the level of BDNF in the serum of the TN group was significantly higher than that in the sham group(P<0.05).The levels of BDNF in the TN+80 mg/kg CBZ group,the TN+40 mg/kg CBZ group,and the TN+20 mg/kg CBZ group were lower than those in the TN group(all P<0.05).Spearman correlation analysis showed that the BDNF level in serum was negatively correlated with mechanical pain threshold(r=-0.650,P<0.01). Conclusion:CBZ treatment can inhibit the expression of BDNF and TrkB in the TGs of TN rats,reduce the level of BDNF in serum of TN rats and the phosphorylation of ERK signaling pathway,so as to inhibit TN.The serum level of BDNF can be considered as an indicator for the diagnosis and prognosis of TN.