人正常卵巢组织中成纤维细胞的培养

Human ovarian fibroblasts culture

目的:通过分离、纯化、培养及鉴定,获得典型人卵巢成纤维细胞。方法:采用酶消化+反复贴壁法,获得纯化成纤维细胞,通过形态学观察和免疫细胞化学染色,对所得细胞进行特征性标志及生物学特性的检测,进一步阐述细胞特征,并绘制细胞生长曲线。结果:形态学上,正常卵巢成纤维细胞形态多样,大部分为梭形、条索形,还有三角形、多角形和不规则形等,并具有伪足样外形;其生长潜伏期较长,12～14 d可传代;免疫细胞化学上,波形蛋白(vimentin)表达强阳性,结蛋白(desmin)、成纤维细胞特异性蛋白1(FSP-1)表达弱阳性;在较早期传代的细胞中,CK-7及CK-P呈少量阳性,随着细胞的纯化,其阳性表达渐行减少直至完全消失;肌动蛋白α(α-SMA)部分细胞表达阳性。而卵巢黏液性细胞标志CK-20、血管内皮细胞标志CD31、间充质细胞标志CD90、雌、孕激素受体(ER、PR)以及基质金属蛋白酶(MMP-2、MMP-3、MMP-9)、肿瘤标志物CA125、CA19-9、癌胚抗原(CEA)、成纤维细胞激活蛋白α(FAP-α)、血小板衍生生长因子受体α(PDGFR-α)均阴性。结论:成功获得人卵巢成纤维细胞,为研究卵巢癌及其相关成纤维细胞提供了必要的细胞学工具。

Objective： To obtain the purified human ovarian normal fibroblasts（NFs） preliminarily.Methods： The ovarian tissues were dissociated by 0.25% trypsin,after overgrowing on the bottom of the culture dish,the cells were adhered to the bottom of the dish repeatedly to depurate them.Then the cells were verified according to its morphology under the inverted-microscope and were further confined by a series of biological markers with immunocytochemistry.And its growth cure was observed.Results： The typical shape of ovarian NFs was fusiform,cords like,triangular,polyangular and irregular.Most of them contain Pseudopods-like structure.The NFs showed positive staining for vimentin,fibroblast specific protein-1,desmin and α-smooth muscle actin,negative staining for cytokeratin pan,cytokeratin 7,cytokeratin 20,CD31,CD90,MMP-2,MMP-3,MMP-9,CA125,CA19-9,CEA,fibroblast activation proteinα（FAP-α） and platelet-derived growth factor receptorα（PDGFR-α）.Conclusion： Human ovarian NFs were cultured successfully,which provides a tool for further studies on ovarian cancer and carcinoma associated fibroblasts.