CAJ | 학술논문

背景：体外分离培养获得足够活性良好的种子细胞是构建阴道组织工程的关键。文献报道阴道上皮细胞体外纯化培养和传代较为困难，尤其是体外长期培养犬等大动物的阴道种子细胞尚未见报道。目的：建立体外稳定培养犬阴道上皮细胞和平滑肌细胞方法。方法：获取犬小块阴道组织，机械分离阴道黏膜上皮，Dispase 酶和胰蛋白酶分步消化收集上皮细胞，接种于无血清角化细胞培养液中培养和传代；机械分离阴道平滑肌组织后采用Ⅱ型胶原酶消化获得平滑肌细胞，在含体积分数10%胎牛血清的 DMEM 培养液中连续培养传代。动态观察上皮细胞和平滑肌细胞生长增殖情况，分别采用特异性抗体行细胞免疫化学染色鉴定。结果与结论：原代培养的上皮细胞24-36 h后开始贴壁铺展，四五天后呈对数生长，七八天可达70%融合，为单一的上皮细胞，呈典型铺路石样，未见成纤维细胞混杂。每四五天可传代1次，连续传代六七次，细胞免疫化学染色角蛋白AEl/AE3抗体阳性。平滑肌细胞原代培养24 h后贴壁呈梭形，此后呈对数生长，4 d后融合呈典型的“峰和谷”样，每三四天可传代1次，连续传代七八次，细胞免疫化学染色示α-肌动蛋白染色阳性。结果证实，犬阴道上皮细胞和平滑肌细胞可在体外长期稳定培养，可为体外构建组织工程化阴道提供足够的种子细胞。
배경：체외분리배양획득족구활성량호적충자세포시구건음도조직공정적관건。문헌보도음도상피세포체외순화배양화전대교위곤난，우기시체외장기배양견등대동물적음도충자세포상미견보도。목적：건입체외은정배양견음도상피세포화평활기세포방법。방법：획취견소괴음도조직，궤계분리음도점막상피，Dispase 매화이단백매분보소화수집상피세포，접충우무혈청각화세포배양액중배양화전대；궤계분리음도평활기조직후채용Ⅱ형효원매소화획득평활기세포，재함체적분수10%태우혈청적 DMEM 배양액중련속배양전대。동태관찰상피세포화평활기세포생장증식정황，분별채용특이성항체행세포면역화학염색감정。결과여결론：원대배양적상피세포24-36 h후개시첩벽포전，사오천후정대수생장，칠팔천가체70%융합，위단일적상피세포，정전형포로석양，미견성섬유세포혼잡。매사오천가전대1차，련속전대륙칠차，세포면역화학염색각단백AEl/AE3항체양성。평활기세포원대배양24 h후첩벽정사형，차후정대수생장，4 d후융합정전형적“봉화곡”양，매삼사천가전대1차，련속전대칠팔차，세포면역화학염색시α-기동단백염색양성。결과증실，견음도상피세포화평활기세포가재체외장기은정배양，가위체외구건조직공정화음도제공족구적충자세포。
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.