mtDNA单倍型D4和B4a细胞线粒体功能及其影响衰老发生的机制研究

A Study of the Mitochondrial Function of mtDNA Haplogroup D4 and B4a Cells and the Mechanism of These Haplogroups Effects on the Occurrence of Aging

该文初步分析了两个与衰老发生抑制及促进的线粒体DNA(mitochondrial DNA,mtDNA)单倍型D4和B4a细胞的线粒体功能情况,并对其影响衰老的可能机制进行了探究。通过细胞融合构建两个核背景一致但mtDNA单倍型分别是D4和B4a的胞质杂合细胞。应用RT-PCR方法检测细胞mt DNA拷贝数以及线粒体基因的mRNA水平,氧电极法和非变性梯度聚丙烯酰胺凝胶电泳(blue native polyacrylamide gelelectrophoresis,BN-PAGE)技术检测细胞线粒体氧化呼吸能力及线粒体复合体水平,TMRM染料和DCFH-DA染料法检测细胞线粒体膜电位和活性氧(reactive oxygen species,ROS)生成水平。结果显示,D4单倍型细胞线粒体氧呼吸能力、膜电位水平以及线粒体基因(mt-ND1、mt-7S RNA)转录水平均明显高于B4a单倍型细胞,而ROS生成水平明显降低,mt DNA拷贝数则并无明显差异。该结果表明,在D4单倍型细胞中,通过提高某些线粒体基因的表达,使得线粒体呼吸复合体表达得到提升,继而提高线粒体氧化呼吸功能,降低细胞氧化损伤,从而延缓衰老的发生。

The aim of this study was to analysis the mitochondrial function in mitochondrial DNA（mt DNA） haplogroup D4 and B4a cell lines and to investigate the possible mechanism of aging. Haplogroup D4 was found to be significantly related to resistance to aging and haplogroup B4a was found to be a risk factor for aging. The mt DNA haplogroup D4 and B4a transmitochondrial cybrids with the same nuclear background were obtained bycytoplasmic hybrid. RT-PCR, clark electrode and gradient blue native polyacrylamide gelelectrophoresis（BNPAGE）, TMRM and DCFH-DA detained, were carried out to examine mt DNA content and mt DNA transcription level, the oxygen consumption rate and mitochondrial complex expression, the mitochondrial membrane potential and reactive oxygen species（ROS） generation, in those cells. Results showed that the mitochondrial oxygen consumption rate, mitochondrial membrane potential and mt DNA（ND1, 7SRNA） transcription level of haplogroup D4 cybrids cell were significantly higher than those of haplogroup B4a cybrids cell, but ROS generation level was significantly lower than that of haplogroup B4a cybrids cell, although there are no significant on mt DNA content level between the two cells. In addition, the mitochondrial function was improved and oxidative damage was decreased in haplogroup D4 cybrids cell by promoting the expression of mt DNA and mitochondrial respiratory complex, so as to delay the occurrence of aging.