| 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66.73 nmol/（mg protein·min）降为36.01 nmol/（mg protein·min）；衰老细胞线粒体蛋白含量（0.78±0.02 mg/ml）高于年轻细胞（0.56±0.03 mg/ml）；以核18S rDNA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。 |
| 关键词: WI-38细胞 线粒体 DNA,线粒体 衰老 |
| DOI：10.3724/SP.J.1008.2006.01290 |
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| 基金项目: |
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| Young and aging human embryonic lung diploid cell line WI-38: a comparison of mitochondrial content, relative amount of mtDNA, and mitochondrial functions? |
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| Abstract: |
| 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 mitochondrial 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; mitochondrial membrane potential was measured by flow cytometry; and NADH oxidase activity was measured by spectrophotometry. 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 50%), 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）. Mitochondrial content in the aging cells（\[0.78±0.02\] mg/ml） was higher than that in the young cells（\[0.56±0.03\] 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 mitochondrial 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. |
| Key words: WI-38 cells mitochondria DNA,mitochodrial aging? |
