人HCT116结肠癌细胞系中CD133^+结肠癌干细胞分离及鉴定.docVIP

人HCT116结肠癌细胞系中CD133＋结肠癌干细胞分离及鉴定 陈克力，梁后杰，江恒，陈建芳，裴莉，郑晨宏(第三军医大学西南医院肿瘤科,重庆400038) 摘要：目的：从人结肠癌细胞系（HCT116）中分离鉴定结肠癌干细胞，并观察其体外生物学特性。方法：利用RT-PCR、免疫荧光、流式细胞等技术检测HCT116细胞中CD133的表达情况；利用免疫磁珠分选技术分选CD133＋细胞；采用无血清培养基培养分选后的CD133＋细胞鉴定其体外增殖特性；采用含血清培养基诱导干细胞克隆球分化并采用免疫组化检测CK20表达情况鉴定其分化特性。结果：HCT116细胞系中存在CD133＋细胞，免疫磁珠能有效的分选CD133＋细胞，分选后的阳性含量为91.92%，分选后的阳性细胞具有典型的干细胞的体外增殖、分化特性。结论：CD133＋结肠癌干细胞在体外具有和普通干细胞类似的无限增殖、自我更新和分化能力，同时它们也表达ABCG2等耐药相关蛋白。 关键词:　HCT116；结肠癌干细胞；CD133；分离及鉴定； Isolation and identification of CD133＋ colon cancer stem cells in HCT116 colon cancer cell line CHEN keli, LIANG houjie, JIANG heng, CHEN jianfang, PEI li, ZHENG chenhong(department of oncology, Southwest Hospital , Third Military Medical University , Chongqing 400038 , China) ABSTRACT: Objective To isolate colon cancer stem cells from human colon cancer cell line HCT116 and observe their biological characteristics in vitro. Methods The expression of CD133 in HCT116 cell line was investigated by Immunofluorescence and FlowCytometry. CD133+ cells were isolated with Magnetic beads. And then the CD133+ cells were cultured in serum-free medium for investgation of proliferation in vitro. Stem spheres was induced to differentiate by serum-containing medium and the expression of CK20 was investigated by Immunohistochemistry. Results There were CD133+ cells in HCT116 cell line. CD133+ cells could be enriched by magnetic islation. The percentage of CD133+ cells wasapproximate to 91.92％ after isolation. The colon cancer stem cells isolated by magnetic beads have typital characteristics of stem cells. Conclusion CD133+ cells that cultured in vitro have the abilities for generation, self-renewing and differentiation as well as normal stem cells. They also express drug resistance associated-proteins such as ABCG2. Key Words：HCT116; colon cancer stem cells; CD133; Isolation and identification; 结肠癌是临床最常见的恶性肿瘤和致死因素之一，化疗是治疗晚期和转移性结肠癌的主要治疗手段，虽然近年来新的化疗药物不断涌现，化疗方案不断更新，但是其完全缓解率和长期生存率仍然低下，目前认为这种情况的出现可能和肿瘤内存在少量肿瘤干细胞有关，这种细胞在肿瘤组织充当干细胞的角色，具有无限增生的潜能