细胞线粒体DNA缺失鉴定的Southern杂交方法的建立  

Establishment of Southern blot to identify the depletion of mtDNA of cells

作　　者：何玉琦[1,2] 凌贤龙[1] 张国桥[3] 李诗伟[1] 乐湘华[1] 晏斌[1] 周源[1] 李歆强[1] 

机构地区：[1]第三军医大学新桥医院消化科,重庆400037 [2]解放军531医院内科 [3]成都军区昆明总医院肿瘤科,昆明650032

出　　处：《解放军医学杂志》2010年第11期1351-1353,共3页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(30470865);新桥医院1520基金

摘　　要：目的建立一种简便、快捷、准确检测细胞线粒体DNA缺失的方法。方法将线粒体DNA所编码呼吸链中细胞色素氧化酶(COX)的亚单位COXⅠ和COXⅡ分别作为Southern杂交的分子探针,鉴别细胞株的线粒体DNA是否完全缺失。进行生长营养缺陷鉴定,并从RNA及蛋白水平对结果进行验证。结果 Southern杂交显示,SK-Hep1细胞可见COXⅠ、COXⅡ杂交条带,ρ°SK-Hep1细胞(线粒体缺失细胞)未见杂交条带形成,作为对比试验的生长营养缺陷法、PCR、Northern杂交和Western杂交,所得结果与Southern杂交结果一致。结论成功建立了鉴定细胞线粒体DNA缺失的Southern杂交方法,该方法能简便、快捷、准确地鉴定细胞线粒体DNA缺失。Objective To establish a convenient, rapid and accurate method for identifying the depletion of mitochondrial DNA （mtDNA） of cells. Methods The human hepatocarcinoma cell line SK-Hepl was cultured in the presence of 100 ng/ml ethidium bromide （EtBr） for more than 20 generations （ρ° cells）. The cells （ρ° SK-Hepl） were then maintained in high-carbohydrate medium DMEM supplemented with 10% fetal bovine serum （FBS）, 50μg/ml uridine （Urd） and 100μg/rnl sodium pyruvate （ρ° culture media）, and the mtDNA depletion was confirmed by Southern hybridization. Cytochrome oxidase （COX） subunits COX I and COX II were used as molecular probes, and growth auxotroph method was employed to check the results at both RNA level and protein level. Results The mtDNA contents of ρ° cells were examined by PCR and Southern blotting, the latter was done with equal amounts of extracted total DNA from SK-Hepl and ρ°SK-Hepl cells. The results demonstrated that mtDNA was amplified from SK-Hepl cells, while not from genomic DNAs of ρ° SK-Hepl cells. On the other hand, the β-actin gene, a nuclear DNA control, was amplified in both SK Hepl and ρ° SK-Hepl cells. The results revealed that the ρ°SK-Hepl cells were entirely devoid of mtDNA. With other methods, such as growth auxotroph test, Northern blot and Western blot it was also confirmed that the ρ°SK-Hepl cells had no objective fragments of mtDNA, implying that the ρ° SK-Hepl cells were entirely devoid of mtDNA. Conclusion A Southern blot method to identify mtDNA depletion from cells has been successfully established. Southern blot is a convenient and accurate method to identify mtDNA depletion from cells.

关 键 词：DNA 线粒体 印迹法 DNA 基因缺失 

分 类 号：Q343.6[生物学—遗传学]