免疫磁珠法原代分离培养人子宫内膜癌微血管内皮细胞及鉴定

目的:建立人子宫内膜癌(EC)微血管内皮细胞的原代分离培养方法并研究其体外免疫组化特征.方法:采用免疫磁珠法结合胶原酶消化法及筛网过滤法分离纯化EC微血管内皮细胞,采用含有20％胎牛血清的M199培养.通过光镜、细胞免疫荧光技术对所获得的微血管内皮细胞进行鉴定,流式细胞仪检测微血管内皮细胞的纯度.微血管内皮细胞的生长曲线通过MTT法测定.结果:微血管内皮细胞台盼蓝拒染率为90％,纯度95％,并能连续传代.原代细胞7～9 d完全融合,传代培养后5～6 d可传代1次.细胞单层生长,融合后呈铺路石样具有接触抑制现象.免疫荧光证实,传代细胞表达内皮细胞特异性vWF因子及细胞间黏附蛋白CD31.经MTT法测定得到的微血管内皮细胞体外生长曲线呈“S”型.结论:成功建立了人EC微血管内皮细胞的分离培养方法,所获得的细胞可用于EC的相关研究,为探讨EC的临床治疗方法提供了新的平台.

Primary culture and characterization of microvascular endothelial cells from endometrial carcinoma with immuomagnetic beads

OBJECTIVE: To establish the method for primary isolating and culturing microvascular endothelial cells of endometrial carcinoma,and to investigate their immunohistochemical characteristics in vitro.METHODS: Using immuomagnetic beads sticking combined with traditional collagenase digestion and filtration,microvascular endothelial cells of endometrial carcinoma were isolated and then cultured on M199 containing 20% fetal bovine serum.The cells were identified by microscopy and immunocytochemistry,and their pureness were detected by flow cytometry.The growth curve of microvascular endothelial cells was assessed by MTT assay.RESULTS: The purity of microvascular endothelial cells which could be passaged serially was over 95%,these cells by trypan blue exclusion dye uptake was 90%.Primary cultured cells were fully integrated 7-9 d in generally,subculture of the cells about 5-6 d could be passaged once.These monolayer cells were fusion with cobbleston-like and they had contact inhibition phenomenon.These cells expressed von willebrand factor and the cell adhesion protein CD31.These cells growth curve were obtained by the MTT assay in vitro showing the "S" shape.CONCLUSIONS: A practical method is established for isolating microvascular endothelial cells of endometrial carcinoma,which can be useful for the studies of endometrial carcinoma.These cultures will be highly relevant to the studies of the clinical endometrial carcinoma treatment.