人HCT116结肠癌细胞系中CD133^+结肠癌干细胞分离及鉴定

Isolation and identification of CD133^+ colon cancer stem cells from colon cancer cell line HCT116

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。

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 cells was investigated by immunofluorescence and flow cytometry, they were isolated with magnetic beads, then they were cultured in serum-free medium for their proliferation in vitro. Stem sphere was induced to differentiate by serumcontaining medium and the expression of CK20 was investigated by immunohistochemistry. The mRNA expressions of CD133, MDR1 and ABCG2 were detected by RT-PCR. Results A few CD133^ ＋ cells existed in HCT116 cells. CD133 ^＋ cells could be enriched by magnetic isolation and had typical characteristics of stem cells. The percentage of CD133^＋ cells after isolation was 91.92%. And those cells expressed MDR1 and ABCG2. Conclusion CD133 ^＋ cells that cultured in vitro possess the capabilities of generation, self-renewing and differentiation as normal stem cells. They also express drug resistance associated-proteins such as ABCG2.