肝癌细胞系Hep3B中CD133^+细胞亚群的分离及其特性的研究

Isolation of CD133^+ Cell and Stem Cell Property from Hepatocarcinoma Hep3B Cell Line

目的研究CD133在人肝癌细胞系Hep3B中的表达以及CD133^+细胞的体外增殖、自我更新及体内成瘤能力,初步探讨肝癌中CD133^+细胞亚群的干细胞特性。方法流式细胞仪检测未分选的Hep3B细胞中CD133^+细胞表达情况;免疫磁珠分选技术纯化CD133^+肿瘤细胞;MTT法检测CD133^+细胞体外增殖能力;无血清培养纯化的CD133^+肿瘤细胞,观察其生长分化情况及非肥胖糖尿病/重症联合免疫缺陷(NOD/SCID)接种实验观察其体内成瘤能力。结果流式细胞仪检测未分选的Hep3B细胞中有9.13%的细胞呈CD133^+表达;免疫磁珠富集的CD133^+肿瘤细胞在无血清培养基中悬浮生长,并可以形成肿瘤细胞球,且在1,3,5,7天的紫外吸光度(A)值均高于相同条件下未分选细胞及CD133^-细胞(P<0.05);CD133^+肿瘤细胞的体内成瘤能力明显高于CD133^-细胞及未分选细胞。结论肝癌细胞系Hep3B中CD133^+细胞亚群比其他细胞亚群具有较强的增殖,分化和成瘤能力,是具有肝癌干细胞特性的细胞亚群。

Objective To detect the expression of CD133 in hepatocellular carcinoma Hep3 B cell line, detect proliferative and self-renew ability of CD133 ＋ tumor cells in vitro, implantation experiment of CD133 ＋ 3 B cell in vivio, and study its stem-cell property. Methods Flow eytometry was used to detect the expression of putative tumor-initiated cell marker CD133 in Hep3B cell line. The isolation technique with immunomagnetie beads was applied to purify CD133 ＋ cells. The proliferative ability of CD133 ＋ tumor cells was estimated by MTY assay in vitro. The proliferative ability of CD133 ＋ , CD133 - and control Hep3B cells were statistically compared. CD133 ＋ tumor cells were cultured in serum-free medium after isolation and observed. Tumor cells implantation experiments were done to test the tumorigenicity of CD133 ＊ , CD133 - and control Hep3B cells. Results 9.13% CD133 ＋ cells were detected in Hep3B cell line. In serum-free RPMI 1640, their UV opti- cal of the CD133 ＋ tumor cells was 0. 311,0. 504,0. 602 and 0. 800 at 1,3,5 and 7 days respectively. Compared with CD133 - cells and control Hep3B cells, CD133 ＋ cells demonstrated increased proliferation capacity. Tumor cell implantation experiments showed that only 1×10^3 CD133 ＋ cells were sufficient for tumor formation,whereas an injection of 1×10^5 CD133 - cells could initiate tumors. Conclusion CD133 ＋ cells subpopulation has stronger ability to proliferate in vitro and to form tumors in vivo than other subpopulations in Hep3B cell line. In general, CD133 ＋ cell subpopulation has stem-cell property.