HuH7细胞系CD13~+CD133~+HCC细胞分选及CSCs特性分析

目的探讨CD13、CD133双荧光标志流式细胞术(fluorescence activated cell sorter,FACS)分选人肝癌HuH7细胞系CD13+CD133+HCC细胞方法及癌干细胞(cancer stem cells,CSCs)特征。方法 CD13、CD133双标志,采用FACS从人肝癌HuH7细胞系分选出CD13+CD133+HCC、CD13+CD133-HCC、CD13-CD133+HCC、CD13-CD133-HCC等4种细胞亚群;通过CD13+CD133+HCC细胞生长曲线,细胞周期,形成肿瘤球和裸鼠体内成瘤能力,及对5-FU、吡柔比星化疗药物的敏感性研究,分析CD13+CD133+HCC细胞是否具有CSCs生物学特征。结果人肝癌HuH7细胞系CD13+CD133+HCC细胞占23.8%,3.1%分选为CD13+CD133+HCC细胞,78.45%的CD13+CD133+HCC细胞处于G0/G1期;CD13+CD133+HCC细胞增殖明显快于其他3个细胞亚群;在裸鼠103个CD13+CD133+HCC细胞就可以成瘤,而1.0×105个CD13-CD133-HCC只有2只成瘤(2/5),干细胞培养CD13+CD133+HCC细胞8～15 d形成肿瘤球;CD13+CD133+HCC细胞对5-FU和吡柔比星具有抵抗特性,而其他3个亚群较易于杀灭。结论 CD13、CD133双标志FACS从HuH7分选的CD13+CD133+HCC细胞具有CSCs特征,可能成为HCC治疗靶细胞。

Sorting and cancer stem cell characteristic analysis of CD13+ CD133+ HCC cells in HuH7 cell line

Objective To study the sorting of CD13+CD133+HCC cell fraction in HuH7 cell line by fluorescence activated cell sorter(FACS) based on the expression of both CD13 and CD133 markers,and to analyze the cancer stem cells(CSCs) characteristics of CD13+CD133+HCC cells.Methods The antibodies against CD13 and CD133 were used as CSCs marker to sort CD13+CD133+HCC cells,CD13+CD133-HCC cells,CD13-CD133+HCC cells and CD13-CD133-HCC cells from HuH7 cell line by FACS.The growth curve,phase of cell cycle,sphere formation ability in vitro,tumor formation ability in vivo and susceptibility to 5-FU and pirarubicin of the CD13+CD133+HCC cells were investigated for CSCs identification and characterization of the CD13+CD133+HCC cells.Results The CD13+CD133+HCC cells accounted for 23.8% in HuH7 cell line,and 3.1% of the CD13+CD133+HCC cells was sorted as CSC-like cells.There was 78.45% of the CD13+ CD133+HCC cells existed in G1/G0 phase.The proliferating ability of the CD13+CD133+HCC cells was significantly stronger than that of the other cell subsets.The CD13+CD133+HCC cells could form a tumor in naked mice after 4 weeks with a number of 103,but 1.0×105 of the CD13-CD133-HCC cells were needed to form a tumor.The in vitro sphere formation of the CD13+CD133+HCC cells was observed after 8 to 15 d.The CD13+CD133+HCC cells had the chemo-resistant ability to 5-FU and pirarubicin,but the other cell subsets could be killed easily by these drugs.Conclusion The CD13+CD133+HCC cells sorted from HuH7 cell line by FACS based on the expression of both CD13 and CD133 markers have the characteristics of CSCs,and may be the therapeutic target for human hepatocellular carcinoma.