目的 从人卵巢癌组织中分离并鉴定卵巢癌干细胞.方法 取新鲜卵巢癌组织,以胶原酶和透明质酸酶充分消化,获得单细胞悬液,经红细胞裂解液处理,CD133磁珠孵育进行磁性细胞分选,流式细胞仪验证分选效率.将阳性细胞置于含人表皮生长因子、碱性成纤维细胞生长因子、牛血清白蛋白和人胰岛素的无血清培养基中,待细胞成球状生长后进行免疫荧光、分化实验和成瘤实验,鉴定其干细胞特性.结果 从人卵巢癌组织中成功分离出肿瘤干细胞,流式细胞仪检测其CD133的表达率为82.5％.该细胞在无血清培养基中呈悬浮球状生长,具有自我更新能力、分化潜能和更强的致瘤能力,具备干细胞特性.结论 人卵巢癌组织中存在卵巢癌干细胞,利用CD133磁珠分选得到的肿瘤干细胞具备干细胞的相关特性,可用于后续卵巢癌干细胞生物学特性的研究.%Objective To isolate and identify the cancer stem cells from primary human ovarian cancer tissues.Methods Fresh tumor tissues from five cases of pathologically diagnosed ovarian cancers were taken,minced and then digested with collagenase and hyaluronidase to obtain single cell suspension.The erythrocytes were removed with ACK Lysis buffer.The suspensions were sorted by magnetic activated cell sorting (MACS) using CD133-binding microbeads.Then the sorted CD133 + cells were verified by flow cytometry.The cells were cultured in serum-free medium supplemented with EGF,bFGF,insulin and BSA,and grew into spheroids.Immunofluorescence,differentiation and tumor formation tests of the cells were performed to characterize the properties of cancer stem cells.Results The ovarian cancer stem cells were successfully isolated from primary human ovarian tumors,which formed typical spheroids in serum-free medium and had stronger ability of tumorigenesis.The results of related experiments verified that CD133positive cells owned the properties of cancer stem cells. Conclusions The ovarian cancer stem cells presenting the characteristics of sternness in vitro and in vivo,have been successfully isolated from primary human ovarian tumor tissues by MACS.The isolated ovarian cancer stem cells could be used in future researches of their biological functions.
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