高永军, 曹毅, 袁勇, 张喜兵, 赵宁辉, 徐蔚. 停循环相关脑损伤的大鼠海马线粒体COX蛋白获取及验证[J]. 云南大学学报(自然科学版), 2014, 36(5): 788-794. doi: 10.7540/j.ynu.20140236
引用本文: 高永军, 曹毅, 袁勇, 张喜兵, 赵宁辉, 徐蔚. 停循环相关脑损伤的大鼠海马线粒体COX蛋白获取及验证[J]. 云南大学学报(自然科学版), 2014, 36(5): 788-794. doi: 10.7540/j.ynu.20140236
GAO Yong-jun, CAO Yi, YUAN Yong, ZHANG Xi-bin, ZHAO Ning-hui, XU Wei. The acquisition and verification of the protein cox-Ⅳ of mitochondrial of  rat hippocampus followed by the ischemic and hypoxic injury under circulation arrest in rats[J]. Journal of Yunnan University: Natural Sciences Edition, 2014, 36(5): 788-794. DOI: 10.7540/j.ynu.20140236
Citation: GAO Yong-jun, CAO Yi, YUAN Yong, ZHANG Xi-bin, ZHAO Ning-hui, XU Wei. The acquisition and verification of the protein cox-Ⅳ of mitochondrial of  rat hippocampus followed by the ischemic and hypoxic injury under circulation arrest in rats[J]. Journal of Yunnan University: Natural Sciences Edition, 2014, 36(5): 788-794. DOI: 10.7540/j.ynu.20140236

停循环相关脑损伤的大鼠海马线粒体COX蛋白获取及验证

The acquisition and verification of the protein cox-Ⅳ of mitochondrial of  rat hippocampus followed by the ischemic and hypoxic injury under circulation arrest in rats

  • 摘要: 为了明确不同脑温停循环对大鼠海马线粒体差异蛋白质的影响,探讨深低温停循环的脑保护机制,研究以16G导管和22G留置针分别穿刺颈内静脉和尾动脉,建立大鼠闭胸式体外循环模型.并在该模型下完成常温(35.5~37.5 ℃)和深低温(27 ℃)状态下停循环10 min,提取海马组织并纯化线粒体,电镜超微结构显示线粒体纯度和完整性均良好,可以进行下一步研究.按照蛋白质组学要求取线粒体蛋白质匀浆,SDS-PAGE预实验显示标本符合蛋白质组学的需要.最后进行i-TRAQ标记的蛋白质组学分析,获取7个差异蛋白质(依据表达量1.5倍或0.67倍,p0.05).之后对其中的差异蛋白质细胞色素C氧化酶进行深入研究,免疫荧光染色精确定性表明线粒体和细胞质的COX蛋白在NTCA和DHCA组间的分布是有差异的,Western-blot半定量分析提示3组之间COX蛋白表达量无明显变化(p0.05),由此判断急性期深低温停循环的脑保护作用与其减少线粒体蛋白质COX向胞质的释放有关。

     

    Abstract: We aimed to investigate the effects of differential proteomics of hippocampal mitochondria in different hypothermic circulation arrest,and to discuss the protection to brain with deep hypothermic circulation arrest.The cardiopulmonary bypass model was established by puncture through jugular vein and caudal artery with 16G canal and 22G detained needle respectively.Then we extracted the hippocampus in every groups after finishing circulation arrest 10 minutes at normal brain temperature(35.537.5 ℃,NTCA)group and deep hypothermic brain temperature(27 ℃,DHCA)except the normal contract group(sham group,35.537.5 ℃,NC).The hippocampal mitochondria was separated and observed by electron microscope,the results showed that the purity and integrity of mitochondria were high and the sample was suited to next experiments.The preliminary experiment of SDS-PAGE showed that the sample was qualified to analysis of isobaric tags for relative and absolute quantification(i-TRAQ) and liquid chromatography-mass spectrometry (LC-MS/MS).We screened 7 differential protein (the changes of expression dose 1.5 or 0.67;p0.05),and then made the further study about one of the differential protein-cytochrome C oxidase.The semi-quantitative study by Western-blot to the sample of hippocampus slice showed no changes in dose totally(p0.05)among 3 groups,at the same time the qualitation study revealed different translocation of COX protein from mitochondria to cytoplasm between NTCA group and DHCA group.So we concluded that DHCA could mitigate hypoxic-ischemic brain damage was relevant with the release of cytochrome C oxidase in hippocampal mitochondria from mitochondria to cytoplasm in acute stage.

     

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