结肠腺癌中结肠癌干细胞的分离、鉴定

Isolation and Identification of EpCAM^(high)CD44^+ Colon Cancer Stem Cells from Colon Adenocarcinoma

目的从人结肠腺癌组织中分离、鉴定结肠癌干细胞,并初步观察其生物学特性。方法利用新鲜结肠腺癌组织,无血清悬浮成球培养,流式细胞检测ESA、CD44表达情况,体外观察其诱导分化及CK20、Muc表这情况,Balb/C小鼠移植观察其成瘤情况。结果从人原代结肠腺癌中分离、纯化EpCAM^(high)CD44^+结肠癌干细胞,结肠癌原代细胞中EpCAM^(high)CD44^+细胞比例为1.7%～38%(平均5.4%)。单克隆形成实验证实结肠癌组织中存在肿瘤干细胞,其比例为(2.07±0.11)%,分离获得的EpCAM^(high)CD44^+细胞能在无血清培养基中"成球",在血清诱导下能贴壁分化;将EpCAM^(high)CD44^+细胞移植在Balb/C裸鼠体内,表现出很强的致瘤性,移植瘤中EpCAM^(high)CD44^+细胞比例为3.6%～43.2%(平均15.2%),所有的移植瘤经组织学测定,均形成腺管样结构,表达结肠特异性分化标志物CK-20、中性上皮粘蛋白(neutral epithelial mucins,Muc)。结论人结肠腺癌组织中存在EpCAM^(high)CD44^+细胞群,具有和普通干细胞相类似的无限增殖、自我更新和分化能力。

Objective To isolate colon cancer stem cells from human fresh colon adenocarcinoma obtained at surgical operation and to study their biological characteristics. Methods colon adenocarcinoma tissues obtained from surgically resected specimens of colon cancer patients are fully chopped, trypsinized, and filtered to prepare single cell suspensions. The cells are cultured in serum-free DMEM/F12 medium, with LIF, EGF, bFGF and 2% fetal bovine serum weekly. The stem cell-markers ESA, CD44 on the isolated cells are detected by using flow cytometry. Tumor stem ceils are induced to differentiate with 10% FBS and expression of cell differentiation markers such as CK20 and Muc. The tumorigenic potent of the isolated cells is evaluated via the transplantation into Balb/C nude mice. Results EpCAM^high CD44^＋ colon cancer stem cells（CCSCs） are isolated and purified from the human primary colon adenocarcinoma. The percentage of CD44 ^＋ EpCAM^high CCSCs in primary colon cancer is 1.7% - 38% （ mean 5.4% ）. There are colon cancer stem cells in colon cancer by limited dilution assay. The percentage is（ 2.07 ＋ 0. 11 ）%. The suspension spheres are generated by the isolated EpCAM^high CD44^＋ CCSCs in serum-free medium. Their differentiation are induced in medium containing 10% FBS. EpCAM^high CD44^＋ CCSCs are implanted＇into Balb/C nude mice, and observed stronger capacity of tumor initiation. The percentage of EpCAM^high CD44^＋ cells in xenografts is 3.6% - 43.2% （ mean 15.2% ）. Human CRC xenografts established in Balb/C nude mice grow as differentiated adenocarcinomas, forming gland-hke structures, and scored positive for expression of colon-specific differentiation markers, such as cytokeratin-20 （ CK20）, and neutral epithelial mucins （Muc）. Conclusion There are colon cancer stem cells in human colon adenocarcinoma. These ceils are characterized by EpCAM^high CD44^＋ surface marker, possessed apparent feature of stem cells and stronger capacity of initiating tumors, and generated spheroid in serum-free medium.