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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。
Objective: To study the expression of mitochondria related genes after ischemia-reperfusion in hippocampal neurons. Methods: The model of Pulsinell four-vessel occlusion was induced by fresh hippocampal tissue extracted from fresh hippocampus tissue after reperfusion for 10 min at 10 min. Reverse transcription followed by liquid hybridization decreased no difference expression sequence. Differentially expressed sequences were amplified by inhibitory PCR. Afterwards, a cDNA library was constructed and positive clones were identified by sequencing. Finally, dot blot was used to confirm differential expression. Results: Seven of the 78 positive clones obtained after construction of the cDNA library were differentially expressed after the final identification. The sequencing tips are expressed fragments of the cytochrome oxidase C subunit and the 4-chain NADH-ubiquinone oxidase complex. CONCLUSIONS: The differential expression of cytochrome oxidase subunit C from another aspect confirms the important role of cytochrome oxidase C in reperfusion injury previously proposed. The expression of NADH - ubiquinone oxidase complex 4 chain after reperfusion injury suggests that the original unknown function of NADH - ubiquinone oxidase complex β subunit may play a role in this pathological process.