论文标题:腺苷A_1受体激动剂预处理延迟相心肌保护作用的蛋白质组学研究
Studies of Proteomics of Adenosine A (1) Receptor Agonist-Induced Delayed Myocardial Protection in Rabbits
论文作者
论文导师 常业恬,论文学位 博士,论文专业 麻醉学
论文单位 中南大学,点击次数 263,论文页数 77页File Size7008K
2007-05-01论文网 http://www.lw23.com/lunwen_1269717/
adenosine A1 receptor; 2-chloro-N6-cyclopentyl-adenosine (CCPA); preconditioning; cardio-protection; late phase; proteomics
研究目的1．研究腺苷A1受体激动剂2-氯环戊腺苷(CCPA)预处理是否对新西兰兔心肌缺血再灌注损伤具有延迟性保护作用；2．用蛋白质组学方法研究CCPA预处理后24h心肌组织蛋白质表达谱的变化。3．用Western印迹法检测其中某些差异蛋白质，寻找可能参与CCPA预处理延迟性心肌保护作用的蛋白质。 研究方法实验分三部分：(1)新西兰大白兔随机分为4组：假手术组(Sham组)、I／R组、NS组和CCPA组。NS组用生理盐水预处理大白兔，CCPA组用100μg／kg CCPA预处理，Sham组及I／R组不给任何预处理，24h后阻断冠状动脉左前降支30min，再灌注120min，Sham组冠状动脉左前降支只套带而不阻断血流，测定心肌梗死面积和血浆心肌酶及肌钙蛋白I浓度，用光学显微镜和电子显微镜观察心肌组织与细胞的结构变化，确定CCPA预处理是否对兔心肌具有延迟性保护作用。(2)用双向凝胶电泳法分离CCPA预处理(CCPA组)和生理盐水预处理(NS组)24h后兔的心肌组织蛋白质，图像分析后找出差异蛋白质，再用基质辅助激光解吸／电离飞行时间质谱(MALDI-TOF-MS)鉴定部分差异蛋白质。(3)根据第二部分结果，用Western印迹法检测其中某些差异蛋白质。 研究结果1．心电图检测显示ST段在缺血期明显抬高，再灌后恢复，说明复制模型过程中缺血和再灌完全，同时血清CK，CK-MB，cTn I浓度显著高于假手术组，说明本研究可以成功建立了新西兰兔心肌缺血再灌注损伤模型；CCPA预处理24小时后心梗面积显著小于I／R组及NS组，血清CK，CK-MB，cTn I浓度明显低于其它各组；光镜及电镜结果显示CCPA组心肌组织细胞损伤减轻。2．双向凝胶电泳分析结果发现表达明显差异的蛋白点有55个，其中表达明显增强(大于3倍)的有25个，表达明显降低(大于3倍)的有16个，表达不匹配(仅在CCPA处理组出现)的有14个。选取其中20个差异蛋白质点进行MALDI-TOF-MS分析，共获得了17张肽质量指纹图谱。并进一步采用Mascot与Expasy软件分析鉴定出了17个蛋白质，这些蛋白包括包括应激反应蛋白(如αB-晶状体蛋白)、代谢相关蛋白(如L-天门冬氨基酸-O甲基转移酶，鸟嘌呤结合蛋白-γ)、信号调节蛋白(如磷酯酰肌醇3激酶)、离子通道蛋白(如电压依赖性阴离子选择性通道蛋白2，接头相关蛋白复合体1)，免疫相关蛋白(HLA-B相关转录本3，MHC-ⅡDR抗原β亚单位，免疫球蛋白重链可变区)等。3．采用免疫印迹技术对其中的一个蛋白质点(232号)代表的αB晶体蛋白进行验证发现，αB晶体蛋白的表达在100μg／kg CCPA处理时亦明显上调。 研究结论1．CCPA预处理对兔心肌具有延迟性保护作用。2．CCPA预处理24h后心肌组织的蛋白质表达谱发生改变：从20个蛋白点中鉴定出17个蛋白质，包括应激反应蛋白(如αB-晶状体蛋白)、代谢相关蛋白(如L-天门冬氨基酸-O甲基转移酶，鸟嘌呤结合蛋白-γ)、信号调节蛋白(如磷酯酰肌醇3激酶)、离子通道蛋白(如电压依赖性阴离子选择性通道蛋白2，接头相关蛋白复合体1)，免疫相关蛋白(HLA-B相关转录本3，MHC-ⅡDR抗原β亚单位，免疫球蛋白重链可变区)等。3．结合Western Blotting鉴定进一步确定CCPA预处理可以诱导αB-晶状体蛋白表达上调；首次发现αB-晶状体蛋白与CCPA预处理延迟性心肌保护作用有关。这为全面揭示CCPA预处理延迟相心肌保护作用的机制提供了重要信息。为临床采用腺苷A1受体激动剂CCPA防治心肌损伤奠定了基础，提供了实验依据。
Objectives: (1) To investigate whether the adenosine A1 receptoragonist 2-chloro-N~6-cyclopentyladenosine(CCPA)could induce the latephase of preconditioning against myocardial ischemia-reperfusion(I/R)injury in rabbits. (2) To investigate the changes of myocardial proteinexpression profiles 24h after CCPA pretreatment, and to search for theproteins probably involved in the late phase of preconditioning inducedby CCPA with proteomics techniques. (3) To identified some differentprotents with western blotting according the above results. Methods: The experiment consisted of three parts: 1.Rabbits weredivided into four groups including group CCPA,group NS,group I/R andgroup sham.CCPA was infused into rabbits in group CCPA,while thesame volume of normal saline(NS)was given those in group NS on day 1.Any treatment was not done in rabbits in groupI/R and sham. 24 hoursafter CCPA or NS prtreatment,rabbits in group CCPA,NS and I/R weresubjected to 30 min left anterior descending coronary artery(LAD)occlusions and were reperfused for 2h. Rabbits in group sham did notsubjecte LAD occulsion and reperfusion. Myocardial infarct size and theconcentrations of cTnI,CK,CK-MB were determined, the tissue and cellinjury of myocardium was examined with optical and electronmicroscope. 2.Proteomic analysis of myocardium of CCPA pretreatment. The left ventricle tissues of CCPA(group CCPA) orNS(group NS)preconditioned rabbits were sampled for proteomicanalysis. 3. According the above results,some protents were identifiedwith western blotting. Results: 1.The concentration of CK, CK-MB, cTn I and themyocardial infarct size were decreased significantly in group CCPA thanthose in group I/R and NS (P＜0.05). The tissue and cell injury ofmyocardium examined with optical and electron microscope in groupCCPA was decreased compared with the group I/R and NS. 2. Theresults revealed that 55 protein spots with significant differentiationexpression were screened. Those protein spots included 25overexpression spots and 16 spots down-regulation in group CCPAagainst group NS, 14 spots only expression in group CCPA. In this study,we selected 20 protein spots to further study by MALDI-TOF-MS andgot 17 peptide mass fingerprints. Furthermore, we identified 17 proteinsfrom those 17 peptide mass fingerprints by using Mascot and Expasybioinformatics software. Those proteins included stress protein (such asαB crystallin), metabolism-associated protein (D-aspartateO-methyltransferase) and signal transduction pathway-related proteins(phosphor- inositide-3-kinase) and so on. 3. we further detected theexpression ofαB crystallin (232 spot) by using immunobltting (WesternBlot). The results suggested thatαB crystallin was up-regulated by the 100μg/kg CCPA pretreatment. Conclusions: 1 Adenosine A1 receptor agonist 2-chloro-N~6-cyclopentyladenosine(CCPA)could induce the late phase ofpreconditioning in rabbits heart. 2. CCPA pretreatment 24h before canresult in the changes of myocardial protein expression profiles.Thedifferential proteins identified by MALDI-TOF-MS might be involved inthe delayed cardioprotection induced by CCPA. It would be helpful forclarifying the mechanisms of CCPA-mediated the second windowprotection against myocardial injury.
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