线粒体DNA损伤与人肝癌细胞多药耐药关系的研究

Study on the relationship between mitochondrial DNA injury and multidrug resistance of human hepatocellular carcinoma

目的:建立线粒体DNA(mitochondrial DNA,mtDNA)缺失肝癌SK-Hep1细胞(ρ°SK-Hep1)的转线粒体模型(cybrid,Cyb),探讨mtDNA缺失诱导人肝癌细胞多药耐药表型产生的可能机制。方法:采用聚乙二醇融合法,将正常人血小板融合入ρ°SK-Hep1细胞,建立ρ°SK-Hep1细胞转线粒体模型SK-Hep1Cyb,并采用PCR、Southern杂交进行鉴定;计数法描绘细胞生长曲线,计算倍增时间;Transwell实验检测细胞侵袭能力,MTT法检测药物敏感性,Western印迹法检测细胞中P-糖蛋白(P-glycoprotein,P-gp)、多药耐药相关蛋白1(multidrug resistance-associated protein 1,MRP1)和Bcl-2的表达水平。结果:PCR、Southern杂交证实ρ°SK-Hep1融合了正常人血小板线粒体,成功建立了转线粒体细胞模型SK-Hep1Cyb。ρ°SK-Hep1细胞群体倍增时间明显缩短(P<0.01),生长速度加快,侵袭能力增强;与SK-Hep1细胞和SK-Hep1Cyb细胞相比,ρ°SK-Hep1细胞对化疗药物耐药性增强,细胞内P-gp、MRP1和Bcl-2蛋白表达增加。结论:采用细胞融合技术成功建立了ρ°SK-Hep1细胞的转线粒体模型。P-gp、MRP1和Bcl-2蛋白表达增加在mtDNA缺失诱导多药耐药表型产生中可能具有一定的作用。

Objective：To establish a DNA-depleted ρ°SK-Hep1 cells＇ transmitochondrial model（cybrid or Cyb）and analyze the influence of depletion of mitochondrial DNA on generation of multidrug resistance phenotype of hepatoma cells and the possible mechanism.Methods：Normal human blood platelets were used as mitochondrial donors,and polyethylene glycol was used as fusion promoting reagent to establish ρ°SK-Hep1 cells＇ transmitochondrial model.These cybrids were identified by PCR and Southern hybridization.Cells growth was recorded by cell counting method and the doubling time was calculated.The ability of cell invasion was detected by Transwell experiment,and drug sensitivity was assessed by MTT assay.Western blotting was applied to detect the expression of P-glycoprotein,multidrug resistance protein 1（MRP1）,and Bcl-2 protein.Results：PCR and Southern hybridization confirmed that cybrids cells had objective fragments of mitochondria of normal human blood platelets and the transmitochondrial model of ρ°SK-Hep1 cells was successfully established.The doubling time of ρ°SK-Hep1 cells was significantly shortened（P〈0.01）,the growth speed was ac-ceralated,and the invasive capability was enhanced.Compared with SK-Hep1 cells and SK-Hep1Cyb cells,ρ°SK-Hep1 cells showed increased resistance to anticancer drugs and elevated expression of P-glycoprotein,MRP1,and Bcl-2 proteins.Conclusion：ρ°SK-Hep1 cells＇ transmitochondrial model was successfully established by using cell fusion technology.Overexpression of P-glycoprotein,MRP1,and Bcl-2 proteins might play a role in the mtDNA depletion induced multidrug resistance of human hepatocellular carcinoma.