正常与脑缺血大鼠脑皮质线粒体的比较蛋白质组学研究

Comparative proteomic analysis on cerebral cortex mitochondria between ischemic and normal cerebral contex in rats

目的采用比较蛋白质组学方法观察正常与脑缺血大鼠脑皮质线粒体蛋白表达差异。方法SD大鼠按随机数字表法分为正常组和缺血组,采用改进的栓线法制备大脑中动脉栓塞(middle cerebral antery occlusion,MCAO)模型,分离大鼠缺血及正常大脑皮层,纯化线粒体蛋白进行二维电泳和凝胶图像分析,对差异蛋白质用HPLC-Chip/MS纳流液质联用技术进行序列分析,经Spectrum Mill搜索NCBInr数据库后鉴定蛋白质。结果正常与脑缺血大鼠脑皮层线粒体蛋白表达存在显著差异,这些差异蛋白经鉴定,主要为TUC-4b、肌酸激酶同工酶、HS1结合蛋白、线粒体核糖体蛋白S27、异柠檬酸脱氢酶、丙酮酸脱氢酶、二氢硫辛酸脱氢酶、泛醌-细胞色素C还原酶、琥珀酸辅酶A还原酶、细胞色素氧化酶、F1-ATP beta链、热休克蛋白70、热休克蛋白60、脂酰辅酶A脱氢酶等差异线粒体蛋白。结论脑缺血可以导致皮层线粒体相关蛋白表达改变,提示其作用可能与线粒体能量代谢、凋亡有关。

Objective To identify the proteins related to ischemia in the mitochondia of cerebral cortex. Methods Totally 7 adult male SD rats were inflicted by middle cerebral artery occlusion （MCAO） for 6 h with nylon monofilament. Another 7 rats served as normal control. Then cerebral cortex were collected and mitochondria were extracted and purified. After 2-dimensional electrophoresis and PDQuest for gel image analysis, the MS/MS of the peptides from the differential proteins digested by tripsin were analyzed by HPLC-Chip/MS and the original data were pre-processed by Spectrum Mill then searched in NCBInr database. Results Specific protein spots were identified as TUC-4b, CKB, HS1 binding protein, mitochondrial ribosomal protein S27, dihydrolipoamide dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase （NAD ＋ ） alpha, dihydroli-poamide S-acetyltransferase, cytochrome c oxidase subunit Vla, succinate-coenzyme A ligase, ubiquinol-cytochrome c reductase core protein, ATP synthase beta subunit, heat shock 70 x 103 protein, heat shock 60 x 103 protein, acyl-coenzyme A dehydrogenase family. Conclusion Cerebebral ischemia affects the proteins related to cerebral cortex mitochondria, which suggests that these proteins may be correlated with the energy metabolism and apoptosis of mitochondria.