目的：建立一种体外有效富集、培养和鉴定具有肝癌干细胞特征的细胞亚群的方法。方法：采用成球培养法利用肿瘤干细胞样细胞（cancer stem cell, CSC）分化培养基对肝癌Huh7细胞进行富集培养，获得的干细胞样细胞于体外进一步扩增获得肝癌干细胞球。流式细胞术检测Huh7干细胞样细胞表面肿瘤干细胞标志物EpCAM、CD90和CD133的表达，平板克隆集落形成实验和裸鼠成瘤实验分别检测Huh7细胞和Huh7干细胞样细胞的克隆集落形成能力、体内成瘤能力。结果：Huh7细胞成球培养3~7 d后即可形成肝癌干细胞样细胞球，获得的干细胞样细胞具有自我更新和增殖能力，其EpCAM阳性细胞比例较Huh7细胞明显增加\[（99.6%±0.31）% vs（0.12%±0.05）%，P<0.01\]，但两种细胞CD90\[(0.11%±0.06)% vs (0.09%±0.07)%, P>0.05\]和CD133\[(0.17%±0.08)% vs (0.15%±0.05)%， P>0.05]表达差异无统计学意义。Huh7干细胞样细胞克隆集落形成数量明显多于Huh7细胞\[（188.67±12.5）vs （79±16.7）个，P<0.01\]；当接种量为5×104个细胞时，与接种Huh7细胞的对照裸鼠相比，接种Huh7干细胞样细胞的裸鼠成瘤时间更短（11 vs 30 d），成瘤率更高（100% vs 16.67%）；接种5×105数量级的细胞时，实验组成瘤体积\[（171.90±10.94）vs（86.39±11.21）mm3, P<0.01\]和瘤体质量\[（2.98±0.82）vs（0.32±0.17）g, P<0.01\]均明显大于对照组。结论：利用成球培养法能够从Huh7肝癌细胞系中富集培养获得Huh7肝癌干细胞，其具有比Huh7细胞更强的成瘤能力。

Objective:To build a method for enrichment, culture and identification of a cell subset which has the characteristics of liver cancer stem cells (CSC) in vivo. Methods: Huh7 liver cancer cells were subjected to enrichment culture in spheric culture condition with cancer stem cell enrichment medium to obtain liver cancer stem cell-like cells. The stem cell-like cells were continuously cultured and proliferated to form liver cancer stem cell sphere in vitro. Flow cytometry assay was performed to detect the expression of the bio-marker moleculars of CSC such as EpCAM (also named CD326), CD90 and CD133. Colony formation assay and tumorigenicity experiments in nude mice were used to detect the colony formation abilities and tumorigenicity of Huh7 cells and Huh7 stem cell-like cells, respectively. Results: After enrichment culture for 3-7 days, Huh7 cells formed cell spheres. The liver cancer stem cell-like sphere-forming cells exhibited self-renewal ability and multiplication capacity. The proportion of EpCAM positive cells was significantly increased compared to Huh7 cells (\[99.6%±0.31\]% vs \[0.12%±0.05\]%，P<0.01). However, there is no significant difference in the expression of CD90 (\[0.11%±0.06\]% vs \[0.09%±0.07\]，P>0.05) and CD 133 (\[0.17%±0.08\]% vs \[0.15%±0.05\]，P>0.05) between the Huh7 cells and sphere-forming cells. The number of colonies formed by enriched sphere-forming cells was significantly more than that formed by Huh7 cells (\[188.67±12.5\] vs \[79±16.7\]，P＜0.01). Compared with the nude mice inoculation of Huh7 cells in the control group，tumor-forming time of nude mice noculation of Huh7 stem cell-like cells in the experimental group was obviously shorter (11 vs 30 d), and showed a higher ratio of tumor formation(100% vs 16.67%) when all the mice of two groups were seeded with 5×104 cells. Both tumor volume (\[171.90±10.94\] vs \[86.39±11.21\] mm3, P＜0.01) and weight (\[2.98±0.82\] vs \[0.32±0.17\]g, P＜001) of nude mice in the experimental group was respectively bigger and heavier than that of the control group when the seeding cell number was 5×105 cells. Conclusion: Liver cancer stem cell-like cells could be enriched and transformed from Huh7 cells with sphere-forming culture method in vitro, and these cells have more powerful tumorigenicity than Huh7 cells do.

国家自然科学基金资助项目（No. 81101634）、四川省科技厅基金资助项目（No.2012SZ0058）、四川省卫生厅科研课题资助项目（No.110472、110478）和成都军区医药科技“十二五”重点课题资助项目（No. B12018）