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

在法医尸检中，窒息死亡是极难诊断的。因此，分子生物学技术是解释死亡原因不可或缺的手段。线粒体既有DNA，也有核内DNA，预计这种变异是在缺氧条件下线粒体DNA发生的。因此，我试图通过检测线粒体呼吸链相关DNA的一种表现，在心肌和脑细胞中对线粒体DNA呼吸链相关结构域进行一般性分析，其多态性与缺氧猝死病例的关系。我用冷冻标本采集了心肌组织样本，用于鉴定窒息或呼吸器官系统紊乱或循环器官系统紊乱引起的急性心脏死亡和缺氧。用微解剖系统对单细胞进行PCR检测。此外，我做了呼吸链相关结构域的直接测序，并分析了线粒体DNA序列以检测snp（单碱基变异）。从窒息到死亡经过较长时间的病例取得了良好的效果，此外，心脏和肾脏的移植效果显著。但是，对于大脑来说，由于对缺氧的敏感性高，可以作为样本使用，但细胞的崩溃时间较早，而且发现了数据偏差。在实时PCR中，两步PCR法对于检测是必不可少的。在将DNA技术应用于法医猝死病例和通过线粒体DNA变化诊断窒息死亡方面做出了贡献。通过这项研究，有可能推动关于线粒体DNA起源的讨论。

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.