分步消化法原代培养鼠阴道黏膜上皮细胞的研究

目的探讨大鼠阴道黏膜上皮细胞的体外培养和扩增技术,为构建组织工程化阴道动物模型提供种子细胞。方法取大鼠阴道全层组织,经Dispase酶和胰酶分步消化后,接种于无血清角化细胞培养液中连续培养,观察细胞形态、体外生长特性和超微结构,绘制生长曲线,免疫组化鉴定。结果原代细胞培养24-36 h后开始贴壁,7-10d约80%融合,呈铺路石样外观,可连续传5-6代;扫描电镜下细胞表面可见微绒毛嵴;角蛋白染色阳性,细胞纯度98%;第五代细胞为正常二倍体核型。结论该方法培养的阴道上皮细胞增殖状态良好,细胞纯度高,扩增迅速,可在较短时间内获得大量细胞用于组织工程学研究。

PRIMARY CULTURE OF RAT VAGINAL EPITHELIAL CELLS USING FRACTIONAL STEP DIGESTION METHOD IN VITRO

Objective To explore a method of culturing and amplifying rat vaginal epithelial cells in vitro,and to provide seed cells for vagina tissue engineering.Methods The isolated rat vaginal tissues were digested by dispase enzyme and trypsin separately,and cultured in Keratinocyte Serum-Free Medium（K-SFM）.We observed cell morphology,proliferation and ultrastructure with optical and electron microscopy,and drew the growth curve.The cells were identified with Anti-P-CK（pan-cytokeratin） by immunohistochemical staining.Results Praimary cells began to adhere after 24-36 hours,and reached 80% confluency after 7-10 days.Vaginal epithelial cells were polygon-like or irregular globular and showed typical appearances of ＂paving stones＂ after confluention under inversted microscopy.The cells could be subculured to 5-6 generations continuously.Microvilli ridge were observed on cell surface with scanning electron microscope.The expression of pan-cytokeratin staining of the cultured cells was positive.Purity of cells was 98%.The fifth generation cells pocessed normal diploid karyotype.Conclusion The culture cells using the fractional step digestion method display better proliferative activity,higher cell purity and shorter growth time.A large number of epithelial cells are rapidly obtained for tissue engineering of the vagina.