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目的:探讨Huh7细胞中边缘群细胞的分选及边缘群细胞在增殖及分裂能力方面与非边缘群细胞的差异,为肝癌的治疗探索新的思路。方法:采用流式细胞仪分选Huh7细胞中的边缘群细胞和非边缘群细胞,用荧光显微镜观察其胞内Hoechst33342荧光染色强度差异;以半定量RT-PCR测定ABCG2 mRNA表达水平的差异;应用免疫荧光染色检测ABCG2蛋白表达的差异并观察其增殖和不对称分裂能力的差异。结果:非维拉帕米处理组边缘群细胞比例约为2.22%,经维拉帕米阻断后边缘群细胞比例减少至0.74%;边缘群细胞中Hoechst33342的荧光强度明显较非边缘群细胞弱,在边缘群细胞中ABCG2 mRNA表达水平是非边缘群细胞的4.32倍(P<0.01),增殖能力是非边缘群细胞的2.7倍(P<0.01),且边缘群细胞能产生ABCG2(+)和ABCG2(-)子代细胞,而非边缘群细胞只能产生ABCG2(-)子代细胞。结论:Huh7细胞中存在少量边缘群细胞(2.22%),其增殖能力较非边缘群细胞强且具有不对称分裂能力(自我更新和定向分化),具有肿瘤干细胞的特性;因此边缘群细胞分选可以作为肿瘤干细胞分离的一种方法,并且在肿瘤的耐药和进展中具有更重要作用,进一步探讨针对Huh7边缘群细胞生物学行为的研究有望开辟肝癌治疗的新思路。
OBJECTIVE: To investigate the differentiation of marginal group cells in Huh7 cells and the differences in proliferation and division ability between marginal population cells and non-marginal group cells, and to explore new ideas for the treatment of liver cancer. Methods: Flow cytometry was used to sort the edge group cells and non-border group cells in Huh7 cells. Fluorescence microscopy was used to observe the intracellular fluorescence intensity of Hoechst33342 staining. The difference of ABCG2 mRNA expression levels was detected by semi-quantitative RT-PCR. Immunofluorescence staining ABCG2 protein expression differences and to observe the differences in proliferation and asymmetric division ability. Results: The ratio of marginal group cells in non-verapamil group was about 2.22%, and the ratio of marginal group cells in verapamil group was reduced to 0.74%. The fluorescence intensity of Hoechst33342 in edge group cells was significantly weaker than that in non-verapamil group (P <0.01), and the proliferative ability of ABCG2 mRNA was 2.7-fold (P <0.01) higher than that of non-marginal group cells in marginal group cells, and ABCG2 (+) and ABCG2 (-) progeny, whereas non-marginal cells produced only ABCG2 (-) progeny. CONCLUSIONS: Huh7 cells have a small number of borderline cells (2.22%) with enhanced proliferative ability and asymmetric ability to split (self-renewal and directional differentiation) compared with non-border cells and have the characteristics of cancer stem cells. Therefore, It can be used as a method of tumor stem cell isolation and plays a more important role in the drug resistance and progression of the tumor. It is further discussed that the research on the biological behavior of Huh7 marginal group is expected to open up a new idea of liver cancer treatment.