【作者】 金丽艳；

【导师】 徐军美；

【作者基本信息】 中南大学 ， 麻醉学， 2010， 博士

【摘要】 研究目的：1.对异氟烷预处理对人缺血再灌注损伤心肌的保护作用进行临床观察。2.采用蛋白质组学方法研究异氟烷预处理对人缺血再灌注损伤心肌蛋白质表达谱的影响,寻找可能参与异氟烷预处理早期相心肌保护作用的蛋白质。3.用免疫印迹法检测其中某些差异表达的蛋白质,以进一步确定这些差异表达蛋白质在异氟烷预处理心肌保护中的作用。研究方法：实验分三部分：(1)择期心肺转流术下行双瓣膜置换术的风湿性心脏联合瓣膜病患者30例,随机分为对照组(n=15)和异氟烷预处理组(异氟烷组,n=15)。异氟烷组在麻醉诱导后吸入1.1%-1.2%(呼气末浓度)异氟烷30 min,洗脱15 min。对照组不吸入异氟烷,其余用药2组无区别。分别于麻醉诱导后吸入异氟烷前(T0)、主动脉开放30min (T1)、关胸后(T2)、主动脉开放6h(T3)、12h(T4)和24h(T5)测量和记录肺动脉导管数据；分别于T0、T1、T3、T4和T5取血测血清肌钙蛋白(cTnI)、肌酸磷酸激酶同工酶(CKMB)浓度。分别于主动脉阻断前(上腔静脉插管时)和主动脉开放后(上腔静脉退管时)取部分右心耳组织,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。(2)用双向凝胶电泳法分离异氟烷组和对照组体外循环后心肌组织蛋白质,经图像分析找出差异表达蛋白质,再用基质辅助激光解吸／电离飞行时间质谱鉴定部分差异表达蛋白质。(3)根据第二部分结果,用免疫印迹法(Western Blot)检测其中某些差异表达蛋白质。研究结果：1.异氟烷组和对照组MABP在Tl-T4时和T0比较降低,PAOP在T2-T5时降低,SVR在Tl-T5时降低,SV、SI在Tl-T5时升高,CO、CI和LVSWI在T1-T5时升高,差异均有统计学意义(P<0.05或P<0.01)。异氟烷组PAOP在T5时和对照组比较降低,SV和SI在T4-T5时升高,CO, CI和LVSWI在T3-T5时升高,差异均有统计学意义(P<0.05或P<0.01)。异氟烷组和对照组cTnI、CKMB基础值之间无显著性差别,异氟烷组T3后cTnI低于对照组,T1后CKMB低于对照组,2组间比较有显著性差异(P<0.05)。光镜与电镜结果显示异氟烷组心肌细胞损伤轻于对照组。2.蛋白质组学结果：2-DE图像显示对照组有417±15个蛋白点,异氟烷组有454±19个蛋白点。经凝胶图像分析找到差异表达蛋白质点20个,基质辅助激光解析-电离飞行时间对差异表达蛋白质进行鉴定,共鉴定出16个蛋白质。其中,在异氟烷组有6个蛋白质表达上调,3个蛋白质表达下调,有1个新合成的蛋白质,但异氟烷组有10个蛋白质未能得到匹配。这些蛋白质包括细胞骨架蛋白(α-肌动蛋白、α-肌球蛋白重链、结蛋白、波形蛋白)、代谢相关蛋白(醛糖还原酶、3-磷酸甘油醛脱氢酶、线粒体ATP酶)、转录相关蛋白(普通转录因子ⅢC、转录因子AP2、锌指蛋白771)、离子通道蛋白(瞬时型阳离子感受器通道)等。3.采用免疫印迹技术对其中a-肌动蛋白(200号)、α-肌球蛋白重链(277号)、波形蛋白(221号)、线粒体ATP酶(47号)进行验证,发现上述四种蛋白质的表达在异氟烷预处理组明显上调。研究结论：1.异氟烷预处理后人心肌组织蛋白质表达谱发生变化,这些差异表达蛋白质可能通过减少细胞骨架破坏、改善能量代谢等发挥异氟烷预处理早期相的心肌保护作用。这是首次发现异氟烷预处理早期相保护作用与α-肌动蛋白、α-肌球蛋白重链、结蛋白、波形蛋白、醛糖还原酶、3-磷酸甘油醛脱氢酶、线粒体ATP酶、普通转录因子ⅢC、转录因子AP2、锌指蛋白771、瞬时型阳离子感受器通道有关。2.结合Western Blot检测,确定异氟烷预处理早期相通过a-肌动蛋白、α-肌球蛋白重链、波形蛋白和线粒体ATP酶参与对人缺血再灌注心肌的保护作用。更多还原

【Abstract】 Objectives:(1) To investigate the protection of Isoflurane preconditioning on myocardial ischemia reperfusion injury in human. (2) To investigate the changes of myocardial protein expression profiles after Isoflurane pretreatment, and to search for the proteins probably involved in the early phase of preconditioning induced by Isoflurane with proteomics techniques. (3)To identify some different proteins with western blot according the above results.Methods:The experiment consists of three parts:(1) Thirty patients undergoing elective cardiac valve replacement with cardiopulmonary by pass (CPB) were randomly assigned to control group (n=15) and isoflurane group (n=15). In the isoflurane group, isoflurane of 1.0 minimum alveolar concentration end-tidal (1.1%～1.2%) was administered for 30 min followed by a 15 min washout period before the CPB. The control group did not inhale isoflurane, and there was no difference in the other drugs in the 2 groups. Pulmonary arterial dates were measured before inhalation isoflurane (T0),30 min after reperfusion(T1), after closing of thorax(T2), and 6h(T3),12h(T4) and 24h(T5) after reperfusion, respectively. Blood samples for serum cTnI and CKMB levels were obtained at To, T1, T3, T4 and T5. Right atria biopsies were collected at the times when inserting catheter into superior vena cava (SVC) and pulling out the catheter from SVC,the tissue and cell injury of myocardium was examined with optical and electron microscope. (2) Proteomic analysis of myocardium of isoflurane pretreatment. The right atria biopsies of isoflurane group or control group were sampled for proteomic analysis. The total proteins were extracted and separated by two dimensional gel electrophoresis(2-DE), and 16 differential expression protein spots were analyzed with matrix-assisted laser desor-ption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS). (3) According the above results, some proteins were identified with western blot.Results (1) Comparing with To, a significant decrease of mean arterial blood pressure (MABP) was detected at T1～T4(P<0.05或P< 0.01), a significant decrease of mean pulmonary arterial pressure(PAOP) was detected at T2～T5 and systemic vascular resistance(SVR) were detected at T1～T5(P<0.05 or P<0.01), stroke volume(SV) and stroke index(SI) at T1～T5, and cardiac output(CO), cardiac index(CI) and left ventricular stroke work index(LVSWI) at T1～T5 increase significantly in both control and isoflurane group(P<0.05或P<0.01); Comparing with control group, PAOP decreased significantly at T5(P<0.05), and SV, SI at T4～T5, CO, CI and LVSWI at T3～T5 increased significantly(P<0.05 or P<0.01) in isoflurane group. There was a significant decrease of cTnI level on T3～T5 and CKMB level on T1～T5 in isoflurane group (P<0.05) when comparing to control group.The tissue and cell injury of myocardium examined with optical and electron microscope in isoflurane group was decreased compared with the control group. (2) Analysis of 2-DE showed that 417±15 protein spots were seen in control group and 454±19 protein spots in isoflurane group, and that the expression of 20 protein spots were different between the two groups.16 protein spots were chosen to do MS analysis, and 16 proteins were preliminarily identified. Six proteins were up-regulated and three proteins were down-regulated and one protein was appeared in isoflurane group, but ten proteins were disappeared in isoflurane group. These proteins can be classified into four functional groups:Include cytoskeletal protein (Alpha actin、Alpha-myosin heavy chain、mutant desmin、vimentin)、metabolism related proteins (aldehyde reductase、glyceraldehyde-3-phosphate dehydrogenase、mitochondrial ATP synthase)、Transcription associated protein (general transcription factor IIIC、transcription factor AP2、zinc finger protein 771)、and ionophorous protein (transient receptor potential cation channel). (3)We further detected the expression of Alpha actin (200 spot)、Alpha-myosin heavy chain(277 spot)、vimentin (221 spot) and mitochondrial ATP synthase (47 spot) by using immunobltting (Western Blot). It was sure that Alpha actin、Alpha-myosin heavy chain、vimentin and mitochondrial ATP synthase were up-regulated by isoflurane preconditioning.Conclusions:(1) Isoflurane pretreatment can result in the changes of protein expression profiles in the myocardium. The differential proteins might function as decreas injury of cytoskeletal proteins and promote the energy metabolism and regulation of genes and proteins of myocardium to confer cardioprotection. Among these differential proteins, we firstly discovered that Alpha actin、Alpha-myosin heavy chain、mutant desmin、vimentin、aldehyde reductase、glyceraldehyde-3-phosphate dehydrogenase、mitochondrial ATP synthase、general transcription factorⅢC、transcription factor AP2、zinc finger protein 771、transient receptor potential cation channel were involved in the isoflurane-induced early phase of preconditioning. (2) It was sured that Alpha actin、Alpha-myosin heavy chain、vimentin and mitochondrial ATP synthase were promoted with early phase of isoflurane preconditioning by using Western Blot.更多还原