人肝癌干细胞生物学行为的实验研究  

Experimental research on the biological behavior of human liver cancer stem cells

作　　者：姜楠[1,2] 阮丹云[3] 李洋[1,2] 汪国营[1,2] 傅斌生[1,2] 汪根树[1,2] 李华[1,2] 张剑[1,2] 杨扬[1,2] 陈规划[1,2] 

机构地区：[1]中山大学附属第三医院肝移植中心 [2]中山大学器官移植研究所 广东省器官移植研究中心,广州510630 [3]中山大学附属第三医院肿瘤内科,广州510630

出　　处：《中华肝脏外科手术学电子杂志》2014年第1期41-44,共4页Chinese Journal of Hepatic Surgery(Electronic Edition)

基　　金：国家重点基础研究发展计划(973计划)(2009CB522404);国家十二五科技重大专项(2012ZX10002017005);国家自然科学基金(81000959);中央高校基本业务费青年教师培育项目(12ykpy47;12ykpy43);广东省科技计划项目(2009B030801007)

摘　　要：目的探讨人肝癌干细胞的生物学行为。方法取对数生长期的人肝癌MHCC97H细胞接种至无血清培养基悬浮培养,获得稳定传代的人肝癌MHCC97H干细胞。分别取人肝癌MHCC97H干细胞微球和人肝癌MHCC97H细胞,应用逆转录聚合酶链反应(RT-PCR)检测细胞标志物分化群(CD)133、CD90、上皮细胞黏附分子(EpCAM)信使核糖核酸(mRNA)。分别进行克隆形成实验、肿瘤细胞侵袭实验(Transwell实验)、裸鼠成瘤实验观察两种细胞克隆形成、侵袭性及成瘤情况。两种细胞实验数据比较采用t或t′检验。结果人肝癌MHCC97H干细胞CD133、CD90、EpCAM mRNA含量分别为32.70±0.20、66.30±0.32、115.40±4.00,人肝癌MHCC97H细胞分别为1.27±0.29、13.60±1.36、1.34±0.40,人肝癌MHCC97H干细胞CD133、CD90、EpCAM mRNA平均含量明显高于人肝癌MHCC97H细胞(t′=117.8,53.97,83.37;P<0.05)。人肝癌MHCC97H干细胞的克隆形成率为(213±10)%,人肝癌MHCC97H细胞为(54±11)%,差异有统计学意义(t=13.11,P<0.05)。通过Transwell实验发现,人肝癌MHCC97H干细胞穿膜细胞数为(587±120)个/视野,人肝癌MHCC97H细胞为(97±13)个/视野,差异有统计学意义(t=6.38,P<0.05)。在裸鼠成瘤实验中,接种2×103个人肝癌MHCC97H干细胞的裸鼠4周可形成皮下移植瘤,成瘤率1/6;2×105个人肝癌MHCC97H干细胞可使全部裸鼠形成皮下移植瘤,成瘤率6/6;至少需要2×105个人肝癌MHCC97H细胞裸鼠才能形成皮下移植瘤,成瘤率2/6。结论与人肝癌MHCC97H细胞相比,人肝癌MHCC97H干细胞具有更强的侵袭性和更高的成瘤率。Objective To study the biological behavior of human liver cancer stem cells. Methods Human liver cancer MHCC97H cells in logarithmic growth phase were inoculated in the serum free medium for suspension cultivation and human liver cancer MHCC97H stem cells with stable propagation were acquired. Human liver cancer MHCC97H stem cell microspheres and human livercancer MHCC97H cells were collected. Messenger ribonucleic acid （mRNA） of cell markers including cluster of differentiation （CD） 133, CD90, epithelial cell adhesion molecule （EpCAM） was detected by reverse transcription polymerase chain reaction （RT-PCR）. Colony formation assay, tumor cell invasion assay （Transwell assay） and nude mouse tumorigenicity assay were performed respectively to observe the colony formation, cell invasion and tumorigenicity of two kinds of cells. The experimental data of two kinds of cells were compared by t test or t＇ test. Results The mean contents of CD133, CD90, EpCAM mRNA in human liver cancer MHCC97H stem cells （32.70±0.20, 66.30±0.32, 115.40±4.00） were significantly higher than those in human liver cancer MHCC97H cells （1.27±0.29, 13.60±1.36, 1.34±0.40） （t＇=117.8, 53.97, 83.37; P〈0.05）. The colony forming efficiency was （213±10）% in human liver cancer MHCC97H stem cells and was （54±11）% in human liver cancer MHCC97H cells, where significant difference was observed （t=-13.11, P〈0.05）. Through the Transwell assay, the membrane permeating cell count was （587±120）/visual fields in human liver cancer MHCC97H stem cells, and was （97±13）/visual fields in human liver cancer MHCC97H cells, where significant difference was observed （t=-6.38, P〈0.05）. In the nude mouse tumorigenicity assay, subcutaneous transplanted tumors were observed in nude mouse 4 weeks after inoculating 2× 103 human liver cancer MHCC97H stem cells and the tumor formation rate was 1/6. Subcutaneous transplanted tumors were observed in all nude mice by inoculating 2× 105 human liver cancer M

关 键 词：肝肿瘤 实验性 干细胞 培养基 无血清 微球体 

分 类 号：R735.7[医药卫生—肿瘤]