BACKGROUND:LL37, the only antimicrobial peptide of the cathelicidin family identified from the human, not only promotes the proliferation of endothelial cel s, but also plays an important role in angiogenesis and re-epithelialization.
OBJECTIVE:To construct a recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene, and to detect the expression and secretion of LL37 after transfected into the canine vaginal epithelial cells.
METHODS:The cDNA encoding LL37 was amplified by PCR. Recombinant adenovirus expression plasmid encoding LL37 and green fluorescent protein (EGFP) was constructed, and identified using restriction endonuclease technology and DNA sequencing method. Adenovirus particles were generated by cotransfecting the 293-packaging cel line. The adenovirus were col ected, amplified and concentrated, and viral titers were determined by end-point dilution assay by applying serial dilutions of the purified viruses to 293 cel s. Primary cultured canine vaginal epithelial cel s were transfected by the recombinant adenovirus. The transfection efficacy was observed by fluorescence microscope, and the cultured supernatant was col ected to determine the expression of LL37 by ELISA method at 1, 2, 3, 5 and 7 days after transfection.
RESULTS AND CONCLUSION:The adenovirus vector GV314-LL37 with the titer of 3×109 pfu/mL was successful y constructed and identified by DNA sequencing methods. Canine vaginal epithelial cel s were successful y isolated and cultured and grew stably. After transfection, vaginal epithelial cel s could express the EGFP and LL37 efficiently in a time-dependent manner detected by fluorescence microscope and ELISA method. The transfection efficacy of EGFP reached to 89%at 72 hours. The level of LL37 in the cel culture supernatant in the transfection group was significantly higher than that in the control group, the highest expression of LL37 was found at 3 days that lasting for 7 days. In conclusion, the recombinant adenovirus overexpressing human antimicrobial peptide LL37 gene is successful y constructed, which can express and secrete LL37 after transfected into canine vaginal epithelial cel s, providing a foundation for constructing the tissue-engineered vagina possessing anti-infection and neovascularization.

0.05).Urethroscopy showed a smooth and intact internal mucosa,wide urethral caliber and normal-appearing urethral tissue in the 2 groups. Conclusions VECM has the same regenerative process as UECM in the replacement of urethral defect.Moreover,VECM has wider source and better elasticity and mechanical properties,so VECM appears to be an ideal material for urethral replacement.

Objective To detect the mouse testicular gene expression pattern differences be-tween spermatogonial stem cell (SSC) proliferative and differential stages and study the molecular reg-ulation mechanism in SSC proliferation and differentiation. Methods With the interval of 24 days, male Kunming mice were injected intraperitoneally with two doses of busulfan (10 mg/kg) to establish spermatogenesis regeneration models. 36 k Mouse Genome Array was used to detect the differential gene expression profiles between the stages of SSC proliferation and differentiation. Bioinforrnsties analysis was conducted in GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Ge-nomes) pathway to describe the potential roles that may play in spermatogonial stern cells behavior regulation. Results Nine hundred and eleven differential expression genes were identified by gene arrays in mice testes, consisting of 608 up-regulated and 303 down-regulated in SSC proliferation stage and SSC differentiation stage. The differential expression genes were classified by their biological process, molecular function and cellular component, respectively. Alterations with statistical signifi-cance (P<0.05)appeared in 84 KEG(;signal pathways, including Notch and Wnt signaling pathways which had been proved to be important for stem cell maintenance. Fifty-six differential expression genes were selected as genes related to stem cells, among which 40 genes were up-regulated, including some stem cell biomarkers(such as Cd9, StraS, hgbl-, Oct4 and Thyl)and some growth factors(such as Fgf2, Pdgfa and Csfl). Conclustion The regulation of SSC proliferation and differentiation involves inmany differentially expressed genes in various signal pathways. This study provides a molecular basis for the elucidation of the molecular mechanism behind self-renewal and differentiation of spermatogonial stem cells.

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.

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.

Objective To establish a long-term culture system for mouse spermatogonial stem cells(SSCs). Methods Testis cells from 4-6 days postpartum male transgenic BALB/C mice were collected by a modified two-step enzymatic digestion method.After three differential adherence selections,the enriched germ cells were finally suspended in StemPro-34 SFM medium supplemented with other nutrients factors and plated on mouse embryonic fibroblast(MEF)feeder layer.20 ng/ml Glial cell line-derived neurotrophic factor,10 ng/ml basic fibroblast growth factor and 200 ng/ml GDNF-family receptor al were added to the serum-free medium to promote SSCs proliferation.Aduh male BALB/C mice,4-5 weeks old,underwent intraperitoneal injection of 40 mg/kg busulfan as recipient mice.Cultured SSCs were also injected into the seminiferous tubules of the left recipient testis through micromanipulator and right testis as self-control.Testes of recipient mice were observed by a fluorescence stereomicroscope and HE stains at 2 months after transplantation. Results By improved digestion method,the vitality of isolated testis cells was more than 98%and the stem cells was enriched about 18.5 fold. 1-2 days after transferred to MEF feeder, the round germ cells started to proliferate and had the shape of paired or aligned undifferentiated spermatogonia connected by cytoplasmic bridges. After 3-4 days, SSCs proliferated continuously and formed typical colonies. SSCs from BALB/c mice could be cultured and passaged in a steady state for 3 months. Cryostat section through the transplanted testis showed that most of seminiferous tubules were filled with germ cells expressing EGFP.HE staining further showed clearly that seminiferous tubules contained complete spermatogenesis.Conclusions SSCs from BALB/c mice could be cultured in an improved culture system for 3 months.The culture system could facilitate understanding the regulatory mechanism that governs SSCs and might provide an opportunity for the cure of infertility.

Objective To investigate the distribution of Human papillomavirus(HPV)genotyping and age in women treated in People′s Hospital of Hubei Province.At the same time,we want to analyse the characteristics of HPV infection.Methods HPV genotyping was detected from the cervical exfoliated cell samples from 7 746 female patients treated in People′s Hospital of Hubei Province from January 6,2016 to January 12,2017.Results There were 1 336 positive cases detected in 7 746 cases of female pa-tients,and the total positive rate was 17.2%.The top eight of HPV subtype infection were 16,52,58,53,81,18,39,56.There were 1 076 cases of single infection,accounting for 80.5%,and the infection of the two types were found in 202 cases,accounting for 15.1%.There were 47 cases of triple infections,accounting for 3.5% and fourfold infection and more were in 11 cases,accounting for 0.9% of all cases.The infection rate of HPV and the infection rate of middle and high risk subtype increased with age,and the infection rate of low risk subtype decreased with age.The detection rate of HPV infection in different age groups had a statistically significant difference(P<0.05).Conclusion The survey of female HPV infection subtype in Hubei Province conforms to the Asian population distribution,and the HPV high-risk subtype infection is mainly distributed in the population over the age of 40.

Objective To investigate the value of detection of human epididymal epithelial secretory protein (HE4) ,carbohy-drate antigen (CA125) and carbohydrate antigen (CA199) in the early diagnosis of ovarian cancer .Methods The clinical data of patients admitted to the hospital from June 2014 to August 2016 were collected from Renmin Hospital of Wuhan University .Ac-cording to the postoperative pathology ,the patients were divided into ovarian cancer group and ovarian benign tumor group .There were 90 cases in ovarian cancer group and 94 cases in ovarian benign tumor group ,98 cases of healthy women in the physical exami-nation center of this hospital were selected as healthy control group .HE4 was detected by ELISA ,serum CA125 and CA199 were detected by chemiluminescence method .Results Compared with healthy control group ,the tumor markers of serum HE4 ,CA125 and CA199 levels were significantly increased (P<0 .01) ,and HE4 and CA125 levels increased more significantly .Compared with ovarian benign tumor group ,the levels of HE4 and CA125 significantly increased in ovarian cancer group ,the difference was statisti-cally significant (P<0 .01) ,and the increase of CA199 less(P<0 .05) .Compared with healthy control group ,the levels of CA125 and CA199 in the benign ovarian tumor group were significantly increased (P<0 .05) .Correlation analysis showed that there were strong correlations between the 3 indexes of HE4 ,CA125 and CA199 in the ovarian cancer group(P<0 .01) .The sensitivity of ser-um CA125 was highest (87 .8% ) in the detection of single marker of ovarian cancer ,while the specificity of serum HE4 was the highest(95 .7% ) .The sensitivity of combined detection of serum HE4 ,CA125 and CA199 was the highest (96 .7% ) ,but the speci-ficity was poor (61 .0% ) .Conclusion Combined detection of serum HE4 ,CA125 and CA199 could significantly improve the early detection rate of ovarian cancer ,but the specificity must be combined with other laboratory tests ,comprehensive analysis and diag-nosis .