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中华肝脏外科手术学电子杂志

中华肝脏外科手术学电子杂志 ›› 2014, Vol. 03 ›› Issue (01) : 41 -45. doi: 10.3877/cma.j.issn.2095-3232.2014.01.011

所属专题： 文献；

基础研究

人肝癌干细胞生物学行为的实验研究
姜楠1,†(), 阮丹云2, 李洋1, 汪国营1, 傅斌生1, 汪根树1, 李华1, 张剑1, 杨扬1, 陈规划1   
  1. 1. 510630　广州，中山大学附属第三医院肝移植中心　中山大学器官移植研究所　广东省器官移植研究中心
    2. 510630　广州，中山大学附属第三医院肿瘤内科
  • 收稿日期:2013-11-16 出版日期:2014-02-10
  • 通信作者: 姜楠
  • 基金资助:
    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(2012ZX10002017-005); 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)

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

Nan Jiang1,†(), Danyun Ruan2, Yang Li1, Guoying Wang1, Binsheng Fu1, Genshu Wang1, Hua Li1, Jian Zhang1, Yang Yang1, Guihua Chen1   

  1. 1. Liver Transplantation Center, the Third Affiliated Hospital of Sun Yat-Sen University, Organ Transplantation Research Institute of Sun Yat-sen University, Organ Transplantation Research Center of Guangdong Province, Guangzhou 510630, China
  • Received:2013-11-16 Published:2014-02-10
  • Corresponding author: Nan Jiang
  • About author:
    Corresponding auther: Jiang Nan, Email:
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姜楠, 阮丹云, 李洋, 汪国营, 傅斌生, 汪根树, 李华, 张剑, 杨扬, 陈规划. 人肝癌干细胞生物学行为的实验研究[J]. 中华肝脏外科手术学电子杂志, 2014, 03(01): 41-45.

Nan Jiang, Danyun Ruan, Yang Li, Guoying Wang, Binsheng Fu, Genshu Wang, Hua Li, Jian Zhang, Yang Yang, Guihua Chen. Experimental research on the biological behavior of human liver cancer stem cells[J]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2014, 03(01): 41-45.

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。

关键词: 肝肿瘤，实验性, 干细胞, 培养基，无血清, 微球体
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 liver cancer 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 MHCC97H stem cells, and the tumor formation rate was 6/6. Subcutaneous transplanted tumors were observed in nude mice by inoculating at least 2×105 human liver cancer MHCC97H cells, and the tumor formation rate was 2/6.

Conclusion

Compared with the human liver cancer MHCC97H cells, human liver cancer MHCC97H stem cells possess stronger invasion and higher tumor formation rate.

Key words: Liver neoplasms, experimental, Stem cells, Culture media, serum-free, Microspheres
图1 无血清悬浮培养法培养7 d获得的人肝癌MHCC97H干细胞球（荧光显微镜×200）
图2 人肝癌MHCC97H干细胞和人肝癌MHCC97H细胞的Transwell实验结果（HE ×200）
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