microRNA慢病毒干扰Rac1基因对糖尿病大鼠脑缺血后线粒体DNA D-loop区基因突变的影响

Incidence of cerebral vascular disease is higher in patients with diabetes mellitus (DM) than that in individuals without DM. Therefore, it is very important to clarify the relationship between DM and cerebral ischemia and to find effective strategies for preventing and treating. Among the hazardous factors, hyperglycaemia has been shown to aggravate brain damage, oxidative stress is one of the important link of disability. Oxidative stress is one of the primary factors that exacerbate damage caused by cerebral ischemia. The maintenance of the mitochondrial genomic integrity is a prerequisite for proper mitochondrial function. Due to the high concentration of reactive oxygen species (ROS) generated by the oxidative phosphorylation pathway, the mitochondrial genome is highly exposed to oxidative stress leading to mitochondrial DNA injury. D-loop region is a hot spot area of the mutations in mitochondrial DNA. It is well known that NADPH oxidase is a major complex that produces detrimental oxygen-derived free radicals during the ischemic period. NADPH oxidase is a multi-component ROS-producing enzyme composed of membrane-bound subunits and cytosolic subunits. Activation of NADPH oxidase is achieved with migration of cytosolic subunits p47phox and p67phox and GTP-Rac1 from cytoplasm to the membrane forming the active NADPH oxidase complex. Rac1, as a member of the Ras superfamily, is known as a key activator of NADPH oxidase. To further define Rac1 function in cerebral ischemic reperfusion injury and to explore novel approaches of gene therapy, we employed the RNA interference technique to knock down gene expression of Rac1 in neuronal cell to study the effect of miRNA by targeting Rac1 gene on the mutation of DNA D-loop region of mitochondrial DNA in diabetic rats in vivo and vitro. We suggest that specific Rac1 inhibition may have potential therapeutic value, and that Rac1 is a novel target for gene therapy of brain ischeamia/reperfusion injury.

糖尿病是引发围术期缺血性脑损伤的独立危险因素，因此阐明糖尿病与脑缺血的关系，寻找有效的防治方法是临床急待解决的棘手课题。在影响缺血性脑损伤的各种危险因素中，高血糖被证明可以加重脑损伤，氧化应激反应是其中一个重要的致残环节。脑缺血缺氧后氧自由基持续不断的积聚，攻击线粒体DNA，引发线粒体 DNA基因突变。线粒体DNA突变的热点区域是D-loop区。在线粒体内调控氧自由基产生的关键酶是NADPH氧化酶，而NADPH氧化酶活性表现为"全或无"的分子基础是其亚基Rac1。因此，本项目通过构建携带有效抑制Rac1基因表达的microRNA的慢病毒感染神经细胞，观察Rac1基因对脑缺血再灌注损伤的保护作用，以及对缺血缺氧后神经细胞线粒体DNA D-loop区基因突变的影响，为围术期缺血性脑损伤治疗，开辟一条新思路，提供一条新的途径。

糖尿病是引发围术期缺血性脑损伤的独立危险因素，因此阐明糖尿病与脑缺血的关系，寻找有效的防治方法是临床急待解决的棘手课题。在影响缺血性脑损伤的各种危险因素中，高血糖被证明可以加重脑损伤，氧化应激反应是其中一个重要的致残环节。脑缺血缺氧后氧自由基持续不断的积聚，攻击线粒体DNA，引发线粒体 DNA基因突变。线粒体DNA突变的热点区域是D-loop区。在线粒体内调控氧自由基产生的关键酶是NADPH氧化酶，而NADPH氧化酶活性表现为“全或无”的分子基础是其亚基Rac1。因此，本项目通过构建携带有效抑制Rac1基因表达的的慢病毒感染神经细胞，观察Rac1基因对脑缺血再灌注损伤的保护作用，以及对缺血缺氧后神经细胞线粒体DNA D-loop区基因突变的影响。.结果显示：.（1）通过神经功能学评分、脑梗死容积百分比测定和HE、Nissl染色评估缺血后的脑损伤情况，我们发现侧脑室注入抑制Rac1基因表达的的慢病毒后或使用Race1特异性抑制剂NSC23766都可以有效减轻糖尿病大鼠脑缺血再灌注损伤。.（2）脑室注入抑制Rac1基因表达的的慢病毒后或使用Rac1特异性抑制剂NSC23766都可以有效减轻抑制Rac1的活化，NADPH氧化酶的活性、ROS的产生以及8-OHdG的激活。.（3）脑室注入抑制Rac1基因表达的的慢病毒后或使用Rac1特异性抑制剂NSC23766都可以促进线粒体内p-Akt、OGG1和POLG的蛋白表达水平的上调。.（4）GenBank中的序列对比，发现是在线粒体DNA D-Loop区第677-678位点之间插入了一个胞嘧啶（C），并且是只有一条DNA序列发生了插入。此突变在侧脑室注入抑制Rac1基因表达的的慢病毒后或使用Rac1特异性抑制剂NSC23766并没有发现，但是具体是否存在功能意义，还需要后续进一步的研究。

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