文章摘要
丁景弦,白小明,张〓海,汪亦品,彭〓韬,冷〓静.Huh7细胞中边缘群细胞分选及其增殖分裂能力的研究[J].南京医科大学学报,2010,(2):139~143
Huh7细胞中边缘群细胞分选及其增殖分裂能力的研究
Studies on the side population cells in Huh7 and their different proliferation and division ability
投稿时间:2009-10-29  
DOI:10.7655
中文关键词: 肝癌  边缘群细胞  维拉帕米  Hoechst33342  ABCG2
英文关键词: hepatocellular carcinoma  side population cells  verapamil  Hoechst33342  ABCG2
基金项目:国家自然科学基金资助项目(30470784,30871015)
作者单位
丁景弦 南京医科大学肿瘤中心,病理学系,生殖医学重点实验室,江苏 南京〓210029 
白小明  
张〓海  
汪亦品  
彭〓韬  
冷〓静  
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中文摘要:
      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?
英文摘要:
      Objective:To investigate the side population cells in Huh7 and their different proliferation and division ability,which may play a key role in the treatment of hepatocellular carcinoma. Methods:Huh7 cells were sorted by fluorescence-activated cell sorter (FACS),and the hoechst33342 fluorescence intensity of side population and main population cells is measured under fluorescence microscope. ABCG2 mRNA levels in side population and main population cells were examined by RT-PCR,which was checked by sequencing. The expression of ABCG2 protein in the clones formed by side population and main population cells was measured by fluorescence immunoassay,and the cell number in ABCG2(+)or ABCG2(-)clones was counted under fluorescence microscope. Results:In the absence of verapamil,the ratio of side population cells was 2.22%,while in the presence of verapamil the ratio decreasesd to 0.74%. Side population cells looked dimmer than main population cells under fluorescence microscope. ABCG2 mRNA level in side population cells was 4.32 times of that in main population cells (P < 0.01). The proliferation ability of side population cells was 2.7 times of that in main population cells (P < 0.01). What’s more,side population cells can differentiate into ABCG2(+)and ABCG2(-)offspring,while main population cells can only differentiate in to ABCG2(-)offspring. Conclusion:Side population cells do exist in Huh7 cell line,and they have stronger proliferation ability compared with main population cells,and asymmetric cell division ability(self-renewal and directed differentiation),which is a key character of stem cells. The isolation of side population cell can be a useful method to isolate cancer stem cell,and these side population cells may play a key role in drug resistance and tumor progression of hepatocellular carcinoma. Further study of the biological behavior of side population cells may pave a new way to the treatment of hepatocellular carcinoma.
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