摘要
目的培养人胚肺二倍体成纤维细胞WI-38,比较年轻和衰老细胞中线粒体量和线粒体DNA(mtDNA)相对含量的变化,以及线粒体功能的改变,探讨线粒体与衰老的关系。方法培养人胚肺二倍体成纤维细胞WI-38;四氮唑盐(MTT)比色法测细胞活力;差速离心分离线粒体,BCA-100Pr定量试剂盒测定线粒体蛋白的含量;竞争PCR法分析mtDNA的相对含量;流式细胞仪测定线粒体膜电位的变化;分光光度法测定线粒体呼吸链氧化酶-NADH氧化酶活性。结果衰老细胞较年轻细胞形成单层时间明显延长,细胞圆缩蜕变,细胞活力明显低于年轻细胞;线粒体膜电位约下降为原来的50%;NADH氧化酶活性也降低,最大反应速度由66.73nmol/(mgprotein·min)降为36.01nmol/(mgprotein·min);衰老细胞线粒体蛋白含量(0.78±0.02mg/ml)高于年轻细胞(0.56±0.03mg/ml);以核18SrDNA为内参,衰老细胞mtDNA相对含量(1.557±0.072)明显高于年轻细胞(1.292±0.068)。结论衰老细胞中,线粒体量和mtDNA相对含量的增高可能是功能下降的一种代偿性反应,为探讨线粒体与衰老的关系提供一定参考。
Objective：To compare the mitochondrial content, the relative amount of mtDNA, and mitochondrial functions between the young and aging WI-38 cells, so as to investigate the correlation between mitochondrial and aging. Methods： Human embryonic lung diploid cell line WI-38 was cultured and its viability was assayed by MTT assay; the content of mitoehondrial protein was determined using BCA-100 Protein Quantitative Analysis Kit after mitochondria were fractionated by differential centrifugation; mtDNA relative content was measured by a competitive polymerase chain reaction （PCR） method; mitoehondrial membrane potential was measured by flow cytometry; and NADH oxidase activity was measured by speetrophotometry. Results： Compared with the young cells, the aging cells had a longer time to form a monolayer, an obviously decreased cell viability and mitochondrial membrane potential （by 50M）, and a decreased NADH oxidase activity, with the maximal reaction speed declining from 66. 73 nmol/（mg protein · min） to 36. 01 nmol/（mg protein · min）. Mitoehondrial content in the aging cells（[0.78±0.02] mg/ml） was higher than that in the young cells（[0.56±0. 031 mg/ml）. Using 18S rDNA of nuclear as an internal reference, the relative amount of mtDNA in the aging cells （1. 557±0. 072） was found to be obviously higher than that in the young cells （1. 292± 0. 068）. Conclusion： The increase of mitoehondrial contents and mtDNA relative amounts in aging cells may be one of the compensatory mechanisms for decreased mitochondrial function, which may provide an evidence for studying the correlation between mitochondrial and aging.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2006年第12期1290-1294,共5页
Academic Journal of Second Military Medical University
基金
国家自然科学基金(30171030
30472175)
中国博士后科学基金(2004036014).~~
