【摘要】：背景:體外分離培養(yǎng)獲得足夠活性良好的種子細胞是構建陰道組織工程的關鍵。文獻報道陰道上皮細胞體外純化培養(yǎng)和傳代較為困難,尤其是體外長期培養(yǎng)犬等大動物的陰道種子細胞尚未見報道。目的:建立體外穩(wěn)定培養(yǎng)犬陰道上皮細胞和平滑肌細胞方法。方法:獲取犬小塊陰道組織,機械分離陰道黏膜上皮,Dispase酶和胰蛋白酶分步消化收集上皮細胞,接種于無血清角化細胞培養(yǎng)液中培養(yǎng)和傳代;機械分離陰道平滑肌組織后采用Ⅱ型膠原酶消化獲得平滑肌細胞,在含體積分數(shù)10%胎牛血清的DMEM培養(yǎng)液中連續(xù)培養(yǎng)傳代。動態(tài)觀察上皮細胞和平滑肌細胞生長增殖情況,分別采用特異性抗體行細胞免疫化學染色鑒定。結果與結論:原代培養(yǎng)的上皮細胞24-36 h后開始貼壁鋪展,四五天后呈對數(shù)生長,七八天可達70%融合,為單一的上皮細胞,呈典型鋪路石樣,未見成纖維細胞混雜。每四五天可傳代1次,連續(xù)傳代六七次,細胞免疫化學染色角蛋白AEl/AE3抗體陽性。平滑肌細胞原代培養(yǎng)24 h后貼壁呈梭形,此后呈對數(shù)生長,4 d后融合呈典型的"峰和谷"樣,每三四天可傳代1次,連續(xù)傳代七八次,細胞免疫化學染色示α-肌動蛋白染色陽性。結果證實,犬陰道上皮細胞和平滑肌細胞可在體外長期穩(wěn)定培養(yǎng),可為體外構建組織工程化陰道提供足夠的種子細胞。
[Abstract]:Background: it is the key to construct vaginal tissue engineering to obtain enough active seed cells in vitro. It is reported in the literature that it is difficult to purify and subculture vaginal epithelial cells in vitro, especially the vaginal seed cells of canine and other large animals have not been reported. Objective: to establish a stable culture method of canine vaginal epithelial cells and smooth muscle cells in vitro. Methods: the canine vaginal tissue was obtained and the vaginal mucosal epithelium was isolated mechanically. The epithelial cells were digested and collected by Dispase enzyme and trypsin respectively. The cells were cultured and subcultured in serum-free keratinocytes culture medium. After mechanical separation of vaginal smooth muscle tissue smooth muscle cells were digested by type 鈪，

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