一种高效实用的人卵巢表面上皮细胞培养方法

An efficient and practical method for culturing human ovarian surface epithelial cells

背景:体外分离培养纯度高、活力强且生物学特性稳定的卵巢表面上皮难度很大,目前原代培养主要采用组织块贴壁法和酶消化法,但这两种方法在取材、细胞活力及细胞纯度上都存在一定的问题。目的:建立一种高效实用的人卵巢表面上皮分离、培养和鉴定方法。方法:创新性地运用细胞刷刷取人卵巢表面上皮,以红细胞裂解法、差速贴壁法对细胞进行分离纯化,并向无血清DMEM-F12培养基中添加人表皮生长因子进行细胞培养。在倒置显微镜下观察细胞形态,应用苏木精-伊红染色和免疫细胞化学染色法对细胞进行鉴定,并绘制生长曲线。结果与结论:卵巢表面上皮培养24h开始贴壁生长,7-12d后基本达到融合,细胞呈多角形或扁平型,透光性及折光性强。细胞形态符合正常上皮细胞特性,所分离的细胞几乎完全表达上皮细胞表面标志物CK19。细胞生长良好,可以传6-8代,细胞生长曲线呈"S"形,纯度达95%以上。结果提示细胞刷取培养法操作简单,能够快速分离获得大量卵巢表面上皮,所获得的细胞经红细胞裂解法和差速贴壁法处理后纯度达95%以上,且细胞生长稳定。

METHODS： The ovarian surface epithelium was obtained with cell brush innovatively, and then the cells were isolated and purified with erythrocyte spallation and differential adherence. The epidermal growth factor was added into the serum-free Dulbecco＇s modified Eagle＇s medium-F12 medium for cell culture. The cell morphology was observed under inverted microscope, and hematoxylin-eosin staining and immunocytochemical staining were used to identify the cells, then the growth curve was draw. RESULTS AND CONCLUSION： The ovarian surface epithelium became adherent after cultured for 24 hours, and reached fusion basically after cultured for 6-12 days. The cells were polygonal or flat with strong transparency and refraction. The morphological characteristics of the cells were in line with those of the normal epithelial cells, and almost all the isolated cells could express the epithelial cells surface marker CK19. The cells could be passaged for 6-8 generations with well growth and the cell growth curve was in ＂S＂ shape. The purity of the cells was more than 95%. The results suggest that cell brush method is simple to operate and can obtain a large amount of ovarian surface epithelium rapidly. The purity of the isolated cells can reach to 95% after treated with erythrocyte spallation and differential adherence method and the cells are in stable growth.