【作者】 李洪；

【导师】 肖颖彬；

【作者基本信息】 第三军医大学 ， 外科学， 2004， 博士

【摘要】 目的:大量的研究证据表明, MitoKATP通道开放在预处理抗心肌梗死中起着非常关键的作用,它是预处理心肌保护的终末效应器。然而最近的研究提示,缺血前MitoKATP通道开放起着启动预处理信号传导的作用。本研究旨在探讨MitoKATP通道开放在预处理心肌保护中的作用及其与ROS生成和PKC epsilon转位激活的相互关系,以及MitoKATP通道开放预处理的功能蛋白质组研究。方法:本课题采用培养成年大鼠心室肌细胞的模拟缺血/再灌注模型和H9C2心肌细胞的缺氧/复氧模型,分三部分进行。第一部分,培养的成年大鼠心室肌细胞随机分成5组,即对照组、缺血/再灌注组、200(mol/L DZ预处理组、200(mol/L DZ+400(mol/L MPG预处理组和200(mol/L DZ+2(mol/L CH预处理组。预处理后的心肌细胞均模拟缺血40min,再灌注30min。用MTT法、CK检测试剂盒、Na+-K+ATPase检测试剂盒和Western blot等方法分别检测细胞的活性、CK的释放、细胞Na+-K+ATPase活性和细胞色素C漏出线粒体的量。用免疫荧光和Western blot方法观察PKC epsilon的转位激活情况。第二部分,培养的H9C2心肌细胞随机分成5组,即对照组、缺血/再灌注组、200(mol/L DZ预处理组、200(mol/L DZ+400(mol/L MPG预处理组和200(mol/L DZ+2(mol/L CH预处理组。预处理后,细胞均被缺氧100 min,复氧30 min。通过标记PI和Hoechst33258以及JC-1,来检测心肌细胞的活性和线粒体膜电位的变化。第三部分,培养的H9C2心肌细胞随机分成2组,即对照组和200(mol/L DZ预处理组,分别富集两组细胞的磷酸化蛋白质,采用双向电泳和质谱技术,分离鉴定DZ预处理后心肌细胞内磷酸化蛋白的差异表达。结果:1. 模拟缺血/再灌注后,各组心肌细胞的活性显著降低 （p<0.01）, 但DZ预处理能课题由国家自然科学基金NO.30200089资助<WP=13>明显增加活细胞数量,与其他预处理组相差显著 （p<0.01）;该心肌保护效应能被MPG和CH消除。2. 模拟缺血/再灌注后, 各组心肌细胞CK的释放量明显增加 （p<0.01）, 但DZ预处理组细胞CK的释放量比较低,与其他预处理组相差显著 （p<0.01）; 而DZ+MPG预处理组和DZ+CH预处理组细胞CK的释放量较DZ预处理组显著增高（p<0.01）;其中以DZ+MPG预处理组增高最明显,与DZ+CH预处理组之间相差显著（p<0.01）。3. 模拟缺血/再灌注后,各组心肌细胞的Na+-K+ATPase活性显著降低（p<0.01）,其中DZ预处理组细胞Na+-K+ATPase活性较高,与其他预处理组相差显著（p<0.01）。4. 模拟缺血/再灌注后,各组心肌细胞明显细胞色素C的释放增加（p<0.01）,但DZ预处理组细胞色素C的释放较少,与DZ+MPG预处理组和缺血/再灌注组相差显著（p<0.01）。 5. 倒置荧光显微镜下观察,正常对照组细胞内可见散在的少数几个点状荧光;DZ预处理组心肌细胞内可见清晰的条状荧光,呈横向排列,与肌丝方向基本一致; DZ+MPG预处理组心肌细胞略有条状荧光;DZ+CH预处理组心肌细胞内分布有少量的点状免疫荧光。Western-blot结果显示,DZ预处理后心肌细胞内的PKC epsilon向膜质部分转位明显（p<0.01）;ROS清除剂MPG和PKC特异性抑制剂CH均能抑制DZ预处理引起的PKC epsilon转位激活（p<0.01）,其中以CH作用最明显（p<0.01）。6. 缺氧/复氧后,各组H9C2心肌细胞PI阳性率显著增加（p<0.01）;DZ预处理组细胞的PI阳性率显著降低（p<0.01）,但该作用可被MPG和CH拮抗。7. 培养的H9C2心肌细胞缺氧/复氧损伤后,各组线粒体膜电位下降明显（p<0.01）;DZ预处理可以升高细胞线粒体膜电位;而当用MPG或CH与DZ共同预处理时,该作用被完全消除。8. 经MALDI-TOF-MS鉴定,差异表达的磷酸化蛋白质有:糖调节蛋白（GRP94）、胞吐相关蛋白重链I、铁蛋白轻链、TCP-1蛋白（CCT） zeta 亚单位和假想蛋白XP₃46548等。结论:1.大鼠心室肌细胞模拟缺血/再灌注损伤模型是研究预处理心肌保护的一种非常有效的细胞模型。2.MitoKATP通道特异性开放剂DZ预处理心肌细胞,对大鼠心室肌细胞模拟缺血/再灌注损伤和H9C2心肌细胞缺氧/复氧损伤都有保护作用,表现在延缓细胞的死亡、减少细胞内CK的释放和线粒体细胞色素C的漏出、增加细胞内Na+-K+ATPase的活性,<WP=14>以及防止线粒体膜电位的降低。3.PKC epsilon参与了MitoKATP通道开放预处理心肌细胞的信号传导,开放MitoKATP通道能引起PKC epsilon由胞浆向肌丝样结构的转位, 它可能是MitoKATP通道的下游信号分子。ROS清除剂能部分阻断PKC epsilon的转位激活,在MitoKATP通道开放预处理的信号传导中,ROS可能PKC epsilon的上游信号分子。4.在MitoKATP通道开放预处理过程中,阻断ROS的生成能消除心肌保护效应。 ROS可能是MitoKATP通道开放预处理心肌保护机制中重要的信号分子,预处理期使用自由基清除剂不利于心肌保护。5.MitoKATP通道特异性开放剂DZ预处理心肌细胞,可能使糖调节蛋白（GRP94）、胞吐相关蛋白重链I、铁蛋白轻链等蛋白发生了去磷酸化修饰,而分子伴侣包含TCP-1蛋白（CCT） zeta 亚单位和假想蛋白XP₃46548等发生磷酸化修饰。这些发生了磷酸化修饰改变的蛋白质可能参与了MitoKATP通道开放预处理心肌保护。更多还原

【Abstract】 Objective A great deal of accumulated evidence implicate opening of mitochondrial ATP-sensitive potassium (MitoKATP) channel as an important step in the anti-infarct effect of ischemic preconditioning, it is the end-effector of preconditioning’s cardioprotection. Recent studies, however, reveal that MitoKATP opening prior to ischemia can actually serve as a trigger of preconditioning’s signal transduction. The purpose of this study was to investigate the mechanisms of cardioprotection about ROS producing and PKC epsiolon translocation and functional proteomics in myocytes preconditioning by MitoKATP channels opener.Methods cultured adult rat ventricular myocytes and H9C2 myocytes were used as experimental ischemia/reperfusion model and hypoxia/reoxygen model. The study was carried out in Three parts: In the first part, cultured adult rat ventricular cardiomyocytes were randomly divided into five groups, including control group, ischemia/reperfusion group, 200(mol/L diazoxide (DZ) preconditioning group, 200(mol/L DZ and 400(mol/L N-(2-mercaptopropionyl) glycine (MPG) , 200(mol/L DZ and 2(mol/L chelerythrine chloride (CH) preconditioning group.After preconditioning, cardiomyocytes were treated with simulated ischemia for 40min and reperfusion for 30 min. The cell viability, creatine kinase released from cell, activity of Na+-K+ATPase and cytochrome C released from mitochondria were respectively measured by MTT methold, CK test kit, Na+-K+ATPase test kit and western blot. The PKC epsilon translocation in cells inducing by preconditioning was assayed by immunofluorescence and western blot. In the second part, cultured H9C2 myocytes were randomly divided into five groups, including control group, hypoxia/reoxygen group, 200(mol/L DZ preconditioning group, 200(mol/L DZ and 400(mol/L MPG, 200(mol/L DZ and 2(mol/L CH preconditioning group. After preconditioning, cardiomyocytes were treated with hypoxia for 100min and reoxygen for 30 min. The cell viability and mitochondrial membrane potential were assayed by labeling cells with both propidium iodide (PI) and Hoechst33258 or JC-1.In the third part, cultured H9C2 myocytes were randomly divided into two groups, the control group and 200(mol/L DZ <WP=9>preconditioning group. The phosphoproteins were enriched from H9C2 myocytes in either control group or 200(mol/L DZ preconditioning group, and isolated by two-dimensional electrophoresis. Finally, the differentially expressed phosphoproteins were identified by MALDI-TOF-MS.Results1. The cell viability in all groups treated with simulated ischemia/reperfusion was decreased significantly (p<0.01); Preconditioning by DZ markedly increased the number of live cells (p<0.01), but this effect of DZ preconditioning could be suppressed by MPG or CH (p<0.01). 2. Creatine kinase released from cardiomyocytes increased significantly in all groups treated with simulated ischemia/reperfusion, but the level of CK was much lower in DZ preconditioning group than in the others (p<0.01), and was much higher in DZ and MPG preconditioning group than in DZ and CH preconditioning group (p<0.01).3. The activity of Na+-K+ATPase in cardiomyocytes was inhibited significantly in all groups treated with simulated ischemia/reperfusion (p<0.01), but it was kept much higher in DZ preconditioning group than in the others (p<0.01).4. Lots of cytochrome C released from mitochondria after undergoing simulated ischemia/reperfusion (p<0.01), DZ preconditioning induced cardiomyocytes to release less amount of cytochrome C to cytosol than DZ and MPG preconditioning (p<0.01).5. Under a fluorescence microscope,the fluorescence of PKC epsilon was located on myofibrillar-like structures, but there were a few green fluorescence spot in cells in control group. The myofibrillar-like fluorescence is very faint in DZ+MPG preconditioning group and disappeared in DZ+CH preconditioning group. The result of western blot showed that PKC epsilon translocated to particulate fraction more significantly in DZ preconditioning group (p<0.01) than that in both DZ+MPG precon更多还原