Top1中毒在脑缺血再灌注后神经元凋亡机制的研究

The increased expression of Reactive Oxygen Species (ROS), as a pathological change in neuron after Cerebral Ischemia-Reperfusion Injury, would lead to Single Stranded Breaks (SSBs) of nervous DNA. Once SSBs accumulating in the nucleus of neuron, the neuron will initiate apoptosis. DNA Enzyme Topoisomerase I (Top1) is one of the most important enzymes during the process of DNA replication and transcription, which usually binding to Single Stranded DNA (ssDNA) to form a short-lived, degradable interim complex called Top1-DNA. Recql5, encoded by Recql5, may combine to RNAPII during transcription and so to prevent the Top1-DNA complex from the collision with transcription bubble. Preliminary study found that Top1 Poisoning would induce neuron apoptosis whether in vivo or in vitro and the the lethal median dose ( LD-50) of Recql5 -/- mice (mutant) lower than wild type. Thus we hypothesize that ROS congregate in large number when neuron suffering Cerebral Ischemia-Reperfusion Injury and lead to Top1 Poisoning during transcription. If the increasing number of SSBs overloaded repair system of neuron, it would induce neuron apoptosis. The experimental subject of our Project is Recql5 -/- mice. As a group of novel molecular targets, Top1 Poisoning and the resulted apoptosis of neuron are studied at both cellular and molecular level, in vivo or in vitro. The purpose of this project is to find a key mechanism involving new in Cerebral Ischemia-Reperfusion Injury after stroke and provide support to the development of potential medicine for treatment of ICVD.

脑缺血再灌注后瞬时产生大量氧自由基（ROS）会导致神经元DNA的单链损伤（SSBs），SSBs大量累积会致其凋亡；神经元在转录中需要Top1的介入，期间形成可裂解的Top1-DNA临时复合物。Recql5基因编码的蛋白质在转录过程中与RNA聚合酶Ⅱ结合能降低转录泡与Top1-DNA发生“碰撞”的几率从而减轻Top1中毒，前期研究中发现在小鼠体内外诱导Top1中毒会导致神经元凋亡，且Recql5-/-小鼠的半数致死量明显低于野生型。我们提出假说：脑缺血再灌注后瞬时生成大量ROS使神经元在转录中发生Top1中毒，导致过多Top1中毒介导的SSBs生成，超出相应修复路径的饱和状态，使SSBs在神经元中大量蓄积，启动神经元凋亡途径最终使其凋亡。本课题拟采用Recql5-/-及野生型小鼠在细胞及活体层面探究脑缺血再灌注后导致神经元凋亡过程中Top1中毒所起的作用，为脑缺血再灌注损伤提供新的治疗靶点。

脑缺血再灌注后瞬时产生大量氧自由基（ROS）会导致神经元DNA的单链损伤（SSBs），SSBs大量累积会致其凋亡；神经元在转录中需要Top1的介入，期间形成可裂解的Top1-DNA临时复合物。Recql5基因编码的蛋白质在转录过程中与RNA聚合酶Ⅱ结合能降低转录泡与Top1-DNA发生“碰撞”的几率从而减轻Top1中毒，前期研究中发现在小鼠体内外诱导Top1中毒会导致神经元凋亡，且Recql5-/-小鼠的半数致死量明显低于野生型。我们提出假说：脑缺血再灌注后瞬时生成大量ROS使神经元在转录中发生Top1中毒，导致过多Top1中毒介导的SSBs生成，超出相应修复路径的饱和状态，使SSBs在神经元中大量蓄积，启动神经元凋亡途径最终使其凋亡。本课题采用了Recql5-/-小鼠在体外构建神经元细胞，在细胞层面评估了Top1中毒与神经元凋亡的关联，并成功构建了MCAO 模型，在活体层面评估了急性缺血性脑卒中缺血再灌注后ROS 导致Top1 中毒与缺血再灌注损伤的关联性，以及Top1 中毒的严重程度。旨在为脑缺血再灌注损伤提供新的治疗靶点。