人肝癌细胞系MHCC97H中侧群细胞初探

Study on side population cells in human hepatoceilular carcinoma cell line 97H

目的从人肝癌细胞系MHCC97H中分选侧群细胞，探索其形态、表型和功能特征。方法利用流式细胞仪从MHCC97H中分选得到侧群细胞，用相差显微镜和透射电镜观察其形态学特点；Western blotting观察其ABCG2表达；用流式细胞仪评估侧群细胞与干细胞基因CD133、CD117的关系；用克隆形成实验和NOD／SCID接种实验分别观察侧群细胞体外增殖和体内成瘤情况。结果侧群细胞体积较小，呈圆形或短梭形；侧群细胞的细胞器如线粒体、内质网等明显少于非侧群细胞；流式检测显示侧群细胞中含有CD133、CD117阳性细胞；Western blotting显示侧群细胞和非侧群细胞中均有ABCG2表达，两者无明显差异；平板克隆形成实验显示SP具有较强的克隆形成能力；体内移植实验显示2000个SP细胞即能在体内形成肿瘤。结论肝癌细胞系MHCC97H中侧群细胞具有肿瘤干细胞样细胞的表型和特性；ABCG2可能不是决定肝癌侧群细胞表型的主要因素。

Objective Our aim is to sort side population cells from human hepatocellular carcinoma cell line 97H and to analysis the characteristic of morphology, phenotype and function of SP. Methods Microstructure and ultrastructure of SP cells were assayed by phase microscope and transmission electron microscopy, respectively, followed by sorting from MHCC97H using Flow Cytome- try. The expression of ABCG2 in SP cells was assayed by Western Blotting. CD133 positive cells and CDl17 positive cells in SP cells were investigated by Flow Cytometry. Colony forming assay was used to measure the ability of SP cell growth and Non-obese diabetic/severe combined immunodeficiency （NOD/SCID） xenograft transplant experiments was performed to assay the ability of initiating tumor for SP cell. Results SP cells had round or short shuttle-like shape and were smaller than nonSP in volume. The numbers of organelle in SP cells were less than that in nonSP. Western blotting demon- strated that the expression of ABCG2 in SP cells was similar to that in nonSP cells. Flow Cytometer analysis showed that CD133 and CDl17 positive cells were present in SP cells. Colony-forming assay showed that SP cells had higher clonogenicity. NOD/SCID xenograft transplant experiments demonstrated that 2000 SP cells were sufficient to form tumor. Conclusion SP cells sorted from MHCC97H have characteristic of cancer stem cell-like phenotype and function. ABCG2 was not a molecular determinant of the SP phenotype in HCC.