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Archive > Volume 20 Issue 6 > 2013,20(6):690-695. DOI:10.3872/j.issn.1007-385X.2013.06.010 Prev Next
Basic Research
Culture and identification of liver cancer stem cell-like Huh7 cells
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Abstract:
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 5×104 cells. Both tumor volume (\[171.90±10.94\] vs \[86.39±11.21\] mm3, P＜0.01) and weight (\[2.98±0.82\] vs \[0.32±0.17\]g, P＜001) of nude mice in the experimental group was respectively bigger and heavier than that of the control group when the seeding cell number was 5×105 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.
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Project Supported:
Project supported by the National Natural Science Foundation of China (No. 81101634), the Scientific Research Foundation of the Science and Technology Bureau of Sichuan Province（No.2012SZ0058）, the Scientific Research Project of the Health Bureau of Sichuan Province（No.110472, 110478）, and the Key Project of Medical Science and Technology of Chengdu Military Region during the 12th Five-Year Plan （No. B12018）
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