人线粒体DNA缺失肝癌细胞株建立

Establishment of Human Hepatocellular Cancer Cell Line Lacking Mitochondrial DNA

人线粒体DNA缺失肝癌细胞株(ρ0SK-Hep1)的建立及鉴定。采用溴化乙锭(EB)诱导,PCR、Southern杂交及选择性培养方法进行鉴定。ρ0SK-Hep1细胞在含EB、无尿嘧啶和丙酮酸的选择培养基中从第1天始细胞逐渐悬浮、肿胀,培养第5天以后大量悬浮死亡,且贴壁疏松,10～12天细胞完全悬浮死亡。在选择性培养基中可以正常生长增殖成单层,有少量悬浮。同期非选择培养的SK-Hep1细胞生长正常。PCR结果显示,细胞色素氧化酶I、II(COXI、COXII)及内参G3PDH在SK-Hep1细胞中均可扩增出相应的条带,ρ0SK-Hep1细胞只见内参条带的形成。Southern杂交结果显示,ρ0SK-Hep1细胞未见COXI、COXII杂交条带形成。经EB诱导后成功地获得了ρ0SK-Hep1细胞株。

To establish a human hepatocellular cancer cell line lacking mitchondrial DNA （mtDNA）. Human hepatocellular cancer cell line SK-Hepl was treated by ethidium bromide （EB）, and to verify the cell line lacking mtDNA, the cells were cultured in media without uridine and pyruvate, and PCR and southern hybridization were used to detect cytochrome oxidase subunit Ⅰ （COXⅠ） and Ⅱ （COXⅡ）. After exposure to EB for 1 day, some SK-Hep1 cells began swelling and suspending, almost all cells died for about 10-12 days when cultured in the media without uridine and pyruvate. When SK-Hepl cells were cultured in the media with EB and uridine and pyruvate for 21 generations, COXⅠ and COXⅡ cannot be amplified, and Southern blot suggested there were not COXⅠ and COXⅡ hybridization bands. Treated By EB, we have successfully established a human hepatoceUular cancer cell line lacking mtDNA （ p^0S K-Hep1）.