NRTIs引起的细胞线粒体DNA缺失的检测及其意义  

Detection of NRTIs-induced mitochondrial DNA depletion using real-time fluorescence quantitative PCR

作　　者：孙玉[1] 张洪海[1] 石英[1] 柳雅立[1] 张彤[1] 吴昊[1] 陈德喜[1] 

机构地区：[1]首都医科大学附属北京佑安医院性病艾滋病实验室,北京100069

出　　处：《中国艾滋病性病》2010年第2期144-148,共5页Chinese Journal of Aids & STD

基　　金：国家科技部"十一五"重大专项(2008ZX10001-007);国家自然科学基金(30870853)~~

摘　　要：目的应用实时荧光定量聚合酶链反应(Real-time fluorescence quantitative PCR)检测核苷类逆转录酶抑制剂(NRTIs)司它夫定(d4T)与齐多夫定(AZT),对HepG2细胞线粒体DNA数量的影响,并探讨其意义。方法用0、3、10、100、200、300μmol/L d4T及AZT处理HepG2细胞2周,应用实时荧光定量PCR检测不同浓度药物作用后线粒体DNA的相对含量。结果成功建立线粒体DNA的定量PCR检测方法。d4T组中,药物浓度为0、3、10、100μmol/L的4个组,细胞线粒体DNA相对含量分别为(96.94±5.77)、(53.73±7.14)、(20.78±3.10)、(1.37±0.29),4个组比较差异有统计学意义(P<0.01);而药物浓度为200及300μmol/L的两个组,细胞均死亡。AZT用药组中,药物浓度为0-300μmol/L的6个组,细胞线粒体DNA相对含量分别为(96.94±5.77)、(108.84±7.80)、(172.56±4.70)、(199.51±10.37)、(158.74±6.64)、(64.06±6.27),差异有统计学意义(P<0.01)。结论实时荧光定量PCR检测细胞线粒体DNA方法切实可行,d4T引起细胞线粒体DNA缺失呈浓度依赖性,AZT对细胞线粒体DNA的影响表现为先增高后降低,其机制有待进一步研究。Objective Using real-time fluorescence quantitative PCR to detect mitochondrial DNA content changes within HepG2 cells induced by d4T and AZT. Methods HepG2 cells were treated with different concentrations（0,3,10,100,200,300μmol/L） of d4T and AZT respectively for two weeks. And then mitochondrial DNA contents were detected by real-time fluorescence quantitative PCR. Results Real-time fluorescence quantitative PCR was set up successfully to detect mitochondrial DNA contents. Mitochondrial DNA relative amounts were 96.94±5.77, 53.73±7.14, 20.78±3.10, 1.37±0.29 respectively with d4T concentrations of 0, 3, 10, 100μmol/L. The differences between groups were significant（P〈0.01）. However, they were 96.94±5.77, 108.84±7.80, 172.56±4.70, 199.51±10.37, 158.74±6.64 and 64.06±6.27 respectively with AZT concentrations of 0, 3, 10, 100, 200, 300μmol/L, and the differences between groups were significant（P〈0.01）. Conclusions It is practicable to detect mitochondrial DNA contents with real-time fluorescence quantitative PCR. It is d4T ,rather than AZT , that causes a dose-dependent mitochondrial DNA depletion within HepG2 cells.

关 键 词：核苷类逆转录酶抑制剂 DNA 线粒体 聚合酶链反应 

分 类 号：R512.91[医药卫生—内科学]