【摘要】 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。更多还原

【Abstract】 BACKGROUND: In vitro culture of sufficient vaginal epithelial cells and smooth muscle cells is the key for vaginal tissue engineering. However, the culture, purification and passage of vaginal epithelial cells in vitro are difficult. Primary culture and passage of vaginal epithelial cells from large animals such as canines has not been reported. OBJECTIVE: To establish a stable method of culturing canine vaginal epithelial cells and smooth muscle cells. METHODS: Vaginal epithelial cells 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 collagenase type Ⅱ; the collected smooth muscle cells were cultured in DMEM culture medium containing 10% fetal bovine serum. The cultured cells were passaged regularly. Cell morphology and proliferation characteristics were observed and cell phenotypes were confirmed by morphology and immunohistochemistry staining. RESULTS AND CONCLUSION: Primary vaginal epithelial cells began to adhere after 24-36 hours, grew logarithmically after 4-5 days, and reached 70% confluence after 7-8 days; the epithelial cells showed a typical cobblestone, with no fibroblasts. Cultured epithelial cells 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 cells adhered and grew after 24 hours. The smooth muscle cells were spindle-shaped and proliferated logarithmically. After 4 days, primary cultured smooth muscle cells were confluent and showed a typical shape of "peaks and valleys", and then the cells 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 cells and smooth muscle cells could have a long-term stable culture and proliferation, to provide adequate seed cells for vaginal tissue engineering.更多还原