人肝癌细胞系Huh-7中CD133^+细胞的分离及鉴定

Isolation and identification of CD133^+ cells from human hepatocellular carcinoma cell lines Huh-7

目的通过表面标志分选法富集人肝癌Huh-7细胞中CD133+细胞,并初步鉴定其特性。方法采用流式细胞分选技术从人肝癌细胞系Huh-7中分选出CD133+细胞,并进行干细胞比例分析;通过对CD133+细胞体外成球能力及增殖能力检测,考察CD133+细胞的自我更新能力;观察CD133+细胞在非肥胖性糖尿病/重度联合免疫缺陷小鼠(NOD/SCID)体内的成瘤情况。结果分选获得的CD133+细胞经无血清培养后阳性比例达90%以上;CD133+细胞体外无血清培养3 d即可成球且生长速度较CD133-细胞快;CD133+细胞在NOD/SCID小鼠体内21 d左右即可形成异种移植瘤。结论 CD133+表面标志物分选方法可以高纯度富集CD133+细胞,利用CD133抗体分选获得的CD133+细胞具有肿瘤干细胞样特性。

Objective To enrich CD133 ＋ cells from human hepatocellular carcinoma cell lines Huh-7 cells through fluores- cence activated cell sorting and identify their biological characteristics. Methods CD133 ＋ cells were sorted by flow cytometry and the percentage of them in cultured Huh-7 cells was analyzed. The self-renewing and sphere-forming ability of CD133 ~ cell were observed by light microscope in vitro in comparison with CD133 - cells. Tumor-forming ability of CD133 ＋ cells was observed by xenografts of them in NOD/SCID mice. Results Flow cytometry analysis indicated that the purity of CD133 ~ subset cells exceeded 90% , CD133 ~ subset cells were verified multipotent with the ability of forming tumor spheres within 3 culture days. And CD133 ＋ subset cells were higher proliferative in vitro and had higher tumorigenitic ability in vivo than those of CD133 - subset cells in mice for 21d. Conclusion CD133 ＋ cells super marker sorting method can enrich CD133 ＋ cells in high purity, and CD133 ＋ cells sorted with CD133 antibody pos- sess the characteristics of tumor stem cells.