多发性骨髓瘤U266细胞株中侧群细胞的生物学特性与耐药性的关系

摘要

BACKGROUND:Studies have shown that tumor stem ceils are the basis and the main cause of tumor recurrence and chemotherapy failure.Therefore,the research on the drug resistance of tumor stem cells has become a hotspot in the field of stem cell research.OBJECTIVE:To analyze the biological characteristics of side population (SP) cells in the U266 cell lines in relation to drug resistance.METHODS:Flow cytometry was used to monitor the percentage of SP cells in the U266 cell lines.Fluorescence-activated cell sorter (FACS) analysis was used to isolate SP and non-SP (NSP) cells from the U266 cell lines.Further analyses of the cell cycle,multidrug resistant protein,methyl cellulose cloning assay of SP cells and NSP cells were performed.Real-time quantitative PCR was used to determine the expression of ABCG2 and MDR1 genes.RESULTS AND CONCLUSION:There were (2.46±0.35)％ SP cells in U266 cells,which were most in the G0 phase.The ratio of G0/G1 in SP cells was (81.50±5.42)％,which was significantly higher than that in NSP cells [(39.85±3.21)％;P ＜0.05].The positive expression rates of P-gp and ABCG2 in SP cells were signficantly higher than those in NSP cells (P ＜0.05).The cloning efficiency of SP cells was significantly higher than that of NSP cells (P ＜ 0.05).The mRNA expression of ABCG2 and MDR1 was also significantly higher in SP cells than in NSP cells (P ＜ 0.05).To conclude,a small subpopulation of the isolates of U266 cell lines belong to tumor stem cell-like SP cell subset,most of which are at the G0 phase.ABCG2 and MDR1 genes are highly expressed in SP cells,which particularly plays a important role in the multidrug resistance of multiple myeloma stem cell lines.%背景:研究表明,肿瘤干细胞是导致肿瘤难以根治,化疗失败和疾病复发的基础和主要原因.因此,针对肿瘤干细胞耐药性的研究成为干细胞领域的研究热点.目的:研究多发性骨髓瘤U266细胞株中侧群细胞的生物学特性及其与耐药性的关系.方法:利用流式细胞仪检测U266细胞株中侧群细胞比例,并采用流式细胞分选方法分离侧群细胞和非侧群细胞,进一步比较2个细胞亚群的细胞周期、耐药蛋白、甲基纤维素克隆生长情况;使用RT-PCR方法检测2个细胞亚群ABCG2、MDR1基因的表达.结果与结论:①U266细胞株中侧群细胞比例为(2.46±0.35)％,绝大多数细胞都处于G0期,GO/G1比例分别达(81.50±5.42)％,高于非侧群细胞(39.85±3.21)％,差异有显著性意义(P＜0.05);②侧群细胞中耐药蛋白P-gp,ABCG2阳性表达率高于非侧群细胞,差异有显著性意义(P＜0.05);③侧群细胞的体外克隆形成能力较强,集落形成率高于非侧群细胞,差异有显著性意义(P＜0.05);④侧群细胞中ABCG2、MDR1基因的表达明显高于非侧群细胞(P＜0.05);⑤结果表明,U266细胞株中存在肿瘤干细胞样侧群细胞亚群,绝大多数侧群细胞处于G0期,其ABCG2、MDR1基因表达明显增强,在骨髓瘤干细胞的耐药性机制形成中发挥重要作用.