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.

ObjectiveTo explore the changes of the frequency and cytokine production of CD19 + CD5 + B cells in Endometriosis before and after treatment.MethodsFlow cytometry and quantitative PCR were used to assess the frequency,cytokine expression of CD19 + CD5 + B cells in the peripheral blood,peritoneal fluids and endometrium biopsies of 35 patients with Endometriosis and 29 patients with myoma of uterus (control subjects).Changes in the CD19+ CD5+ B cell compartment in the peripheral blood were compared before and after treatment.ResultsAll 35 endometriosis patients had higher frequencies(all P ＜0.01 ) of CD19+ CD5 + B cells in the peripheral blood,peritoneal fluids and endometrium biopsies [ (13.1 ± 1.9)％,(12.1 ±2.0)％,(11.7 ± 1.7)％] than 29 control subjects[ (2.9 ±0.8)％,(2.6 ±0.9)％,(2.8 ±1.1 )％ ].CD19 + CD5+ B cells from endometriosis patients expressed higher IFN-γ[ (7.2 ± 1.0) × 103,(7.9±1.3) ×103,(7.4±1.1) ×103 copies] than control subjects [ (1.9±0.7)×103,(2.2±0.8)×103,(2.0 ±0.5) × 103 copies).The endometriosis patients also had higher IL-10 production of CD19 + CD5 + B cells [ (6.4 ±0.9) × 103,(6.8 ± 1.2) × 103,(6.1 ±0.8) × 103 copies] than control subjects [ ( 1.7 ±0.5) × 103,(2.1 ±0.9) × 103,( 1.6 ±0.4) × 103 copies].Compared with control subjects,the endometriosis patients had a clinical response to treatment demomtrated a significant decrease in CD19 + CD5 + B cells in the peripheral blood[ ( 13.1 ± 1.9)％ vs (7.3 ± 1.2)％ ],expressed lower IFN-γ [ (7.2 ± 1.0)×103 copies vs (3.3 ±0.6) × 103 copies],produced less IL-10 [(6.4 ±0.9) × 103 copies vs (3.2 ±0.7) × 103 copies].ConclusionsCD19+ CD5 + B cells might play an important role in the pathogenesis of endometriosis through higher IFN-γand IL-10 expression.