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Research on hypoxic sudden death by analysis of mitochondrial respiratory-chain-related DNA

线粒体呼吸链相关DNA分析对缺氧性猝死的研究

基本信息

批准号：
14570381
负责人：
HONDA Katsuya
金额：
$ 2.24万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

In forensic autopsy, death from suffocation is extremely difficult to diagnose. So, technique of molecular biology is indispensable for elucidation of the cause of death.Mitochondria has DNAs as well as intra-nuclear DNA, in addition, it is expected that the variation is caused in mitochondrial DNA under a hypoxic condition. Therefore, I tried to do general analysis of a respiratory-chain-affiliated-domain of mitochondria DNA, in myocardium and intra-cerebral cells, by the detection of a manifestation of a respiratory chain-affiliated-DNA of mitochondria, in the relation with polymorphism with hypoxic sudden death cases. I collected tissue samples of the myocardium with a frozen specimen for the identification of death from suffocation or a respiratory organs system disorder or acute heart death and hypoxia by a circulatory organ system disorder. PCR were performed using a single cell by micro-dissection-system.In addition, I did a direct sequence of a respiratory-chain-related domain and analyzed a sequence of mitochondria DNA to detect SNPs (single base variation).The good results was obtained in the cases relatively long time was passed from asphyxiation to death, in addition, remarkable results were provided with a heart and a kidney. However, as for a brain, usefulness as a sample is expected because high sensitivity of hypoxia, but collapse of a cell was early, and deviation of data was found.In real time PCR, it became clear that two step of PCR method was indispensable for detections.I made contribution to apply DNA technology to forensic medicine in cases of sudden death and a diagnosis of death from suffocation by a change of mitochondria DNA. By this study, it is possible to push forward discussion about the origin of mitochondrial DNA.

在法医尸检中，窒息死亡极难诊断。因此，分子生物学技术是阐明死亡原因必不可少的技术。线粒体除了核内DNA外，还具有DNA，另外，在缺氧条件下，线粒体DNA的变异被认为是引起的。因此，我试图通过检测线粒体呼吸链相关DNA的表现，在心肌和脑内细胞中对线粒体DNA的呼吸链相关结构域进行一般分析，并与缺氧性猝死病例的多态性相关。我用冷冻标本采集了心肌组织样本，用于鉴定窒息死亡或呼吸器官系统疾病或急性心脏死亡和循环器官系统疾病引起的缺氧。用显微切割系统对单个细胞进行PCR扩增，并对线粒体DNA序列进行单碱基变异（single base variation，SNPs）分析，对窒息至死亡时间较长的病例进行SNPs检测，对心脏和肾脏的SNPs检测结果也较为显著。然而，对于脑，由于缺氧的敏感性高，作为样品的有用性是预期的，但是细胞的崩溃是早期的，并且发现了数据的偏差。在真实的时间PCR中，很明显，两步PCR方法对于检测是必不可少的。我为DNA技术在法医学中应用于猝死病例和通过线粒体DNA的变化诊断窒息死亡做出了贡献。通过这项研究，有可能推进线粒体DNA起源的讨论。