大鼠脑缺血再灌注后线粒体呼吸链相关基因表达

Mitochondria respiratory chain related genes expression in post ischemia/reperfusion rat

目的 :研究海马神经元缺血再灌注后线粒体相关基因的表达。方法 :改良Pulsinell四血管阻断模型致大鼠脑缺血 10min再灌注 2 4h后 ,取新鲜海马组织抽提mRNA。逆转录后经液相杂交递减无差异表达序列。以抑制性PCR方式扩增差异表达序列。之后构建cDNA文库 ,测序鉴定阳性克隆。最后应用斑点杂交确认差异表达。结果 :构建cDNA文库后获得的 78个阳性克隆中有 7个克隆经最后的鉴定具有差异表达性。测序提示为细胞色素氧化酶C亚单位和NADH -泛醌氧化酶复合体 4链的表达性片段。结论 :细胞色素氧化酶C亚单位的差异表达从另一个侧面证实了之前提出的再灌注损伤中细胞色素氧化酶C的重要作用。再灌注损伤后NADH -泛醌氧化酶复合体 4链的表达提示原来功能不明的NADH -泛醌氧化酶复合体 β亚基可能在这一病理过程中发挥一定的作用。

AIM: Apply the newly invented method of suppression subtractive hybridization to scan the mitochondria genes expression of hippocampus neurons in post ischemia/reperfusion rats. METHODS: Decapitate the rats suffering 10 min of whole brain ischemia and 24 h of reperfusion in Pulsinelli’s 4 vessels occlusion ischemia/reperfusion model. Dissociated hippocampus and isolated mRNA. After reversed transcription, use SSH to subtract the common sequences, then use suppressed PCR to amplify the differentially expressed sequences. After cloning and DNA sequencing, the dot hybridization was used to finally identify the differential gene expression. RESULTS: In the 78 positive clones acquired from cloning, 7 clones represent differential gene expression after confirmed by clone PCR, dot blot and DNA sequencing. The DNA sequencing data indicate these clones are ESTs of Cytochrome c oxidase subunit and NADH-ubiquinone oxidoreductase chain 4 gene. CONCLUSION: Cytochrome C oxidase mRNA level changs during brain ischemia /reperfusion. The differential expression of NADH-ubiquinone oxidoreductase chain 4, one of those 13 subunits of subcomplex β, suggests that subcomplex β may have important function associated with pathophysiologic changs in reperfusion injury.