人上皮性卵巢癌组织中肿瘤相关成纤维细胞的培养

Cultivation,purification and identification of human ovarian epithelial carcinoma-associated fibroblasts

目的:通过分离、纯化、培养及鉴定,获得人上皮性卵巢癌组织中肿瘤相关成纤维细胞。方法:采用酶消化法获得原代细胞,再通过酶消化+反复贴壁法,传代获得第4代纯化细胞,并绘制细胞生长曲线,通过形态学观察和免疫细胞化学染色,对所得细胞进行鉴定。结果:获得典型卵巢癌相关成纤维细胞。形态学上,细胞呈长梭形,胞核不明显,细胞大小不一致。其生长潜伏期较肿瘤细胞长,生长相对缓慢。免疫细胞化学上,肿瘤相关成纤维细胞中,成纤维细胞特性蛋白-1、波形蛋白、结蛋白及α-平滑肌动蛋白均表达阳性,而血管内皮细胞、间充质细胞标志均表达阴性。细胞角蛋白在较早传代的细胞中少量表达,通过纯化后基本消失。结论:本方法可获得典型上皮性卵巢癌相关成纤维细胞,为进一步研究该细胞自身生物学特性及其与卵巢癌间的相互作用提供了必要的细胞学基础。

Objective：To isolate,purify,culture and identify the carcinoma-associated fibroblasts（CAFs） from the human epithelial ovarian neoplasm.Methods：The primary ovarian CAFs were obtained from human epithelial ovarian neoplasm and purified by trypsin digestion and adherence repeatedly.The cells were verified according to morphological observation and immunocytochemical staining of certain proteins.Additionally,their proliferation indexes were assayed.Results：The purified ovarian CAFs were obtained successfully.The typical morphology of ovarian CAFs was simple fusiform fiber like cells.The ovarian CAFs exhibited lower proliferation than tumor cells and showed positive staining for fibroblast specific protein-1,vimentin,desmin and especially for α-smooth muscle actin,and negative staining for cytokeratin（broad-spectrum）,cytokeratin 7,cytokeratin 20,CD31 and CD90.Conclusion：The primary cultured CAFs derived from the human ovarian carcinoma tissue can be obtained successfully with digestion method.This provides us an experimental foundation for further studies on the roles of CAFs in the initiation and progression of the cancer.