【作者】 张云仙；

【导师】 贾钰华；

【作者基本信息】 南方医科大学 ， 中西医结合基础， 2008， 硕士

【摘要】 一、目的随着溶栓疗法、经皮冠状动脉腔内成形术、冠状动脉内支架置入术等治疗心肌梗塞的推广应用,急性心肌梗塞时缺血心肌重新得到血液灌注的方法越来越多。实验和临床研究发现心肌缺血后尽早恢复血流供应,能缩小心肌梗塞面积,降低死亡率,但再灌注同时造成心肌细胞损伤,表现为再灌注心律失常、心肌组织结构改变、心肌顿抑等,其中再灌注心律失常是再灌注损伤的主要表现,严重的室性心律失常是致心源性猝死的重要原因。因此,探讨缺血再灌注心律失常的病理机制及研究有效的防治药物意义重大。西医对心律失常的治疗,不论在手术及电子仪器等方面,均有很大的发展,但仍以药物为主。当前治疗心律失常的药物很多,但长期服用都有一定的毒副作用,尤其是抗心律失常的药物导致的心律失常,引起了医药学家的特别重视。中医药防治心律失常具有多层次、多角度、多靶点的特征,并且无明显的毒副作用。本研究采用大鼠缺血再灌注心律失常动物模型,研究中药复方定心方及单体氧化苦参碱对再灌注心律失常的防治作用;研究定心方及氧化苦参碱对再灌注心律失常血清细胞间粘附分子-1(intercelluar adhesionmolecule-1,ICAM-1)表达的影响;同时利用蛋白质组学技术研究再灌注心律失常及定心方和氧化苦参碱干预后心肌蛋白质表达谱的变化,进一步阐述再灌注心律失常的分子机制和定心方及氧化苦参碱抗心律失常的作用机理,为临床上防治再灌注心律失常提供理论依据。二、方法32只雄性Wistar大鼠随机分为4组,每组8只,分别为假手术组(shamoperated group,SH)、再灌注组(ischemia-reperfusion group,I/R)、定心方干预组(DingXin Recipe group,DXR)及氧化苦参碱干预组(Oxymatrine group,OMT)。所有动物均适应性喂养两天。SH组、I/R组和OMT组均灌服生理盐水,DXR组灌服定心方药液,连续灌胃7天后,SH组冠脉穿线但不结扎,I/R、DXR和OMT组手术造模。根据《药理实验方法学》“麻醉大鼠冠脉结扎和再灌注诱发心律失常法”制作模型。连接心电图机和心电示波器,观察心律失常的发生,并详细记录再灌注期间的心律失常,进行室性心律失常的量化评分;腹主动脉取血,分离血清;取心脏,冰生理盐水冲洗,分离左心室,-80℃冰箱保存。左冠脉结扎和再灌注诱发的心律失常以室早(premature ventricularcontraction,VE)、室速(ventricular tachycardia,VT)、室颤(ventricularfibrillation,VF)等室性心律失常(ventricular arrhythmias,VA)为主,故根据Curtis等创立的VA评分方法,进行室性心律失常评分。腹主动脉采血分离的血清,采用双抗体央心ABC-ELISA法检测血清中ICAM-1的表达,观察定心方及氧化苦参碱对再灌注心律失常血清ICAM-1表达的影响。部分心肌组织用组织裂解液提取心肌总蛋白,采用Bradford法进行蛋白定量。定量后采用双向电泳技术分离心肌总蛋白,一向采用IPG3-10L、24cm胶条进行水平的等电聚焦,二向进行SDS-PAGE垂直电泳。电泳结束后用考马斯亮兰进行凝胶染色,后利用Umax PowerLook 1100投射扫描仪采集图像。所获得的图像用Bio-Rad二维图谱分析软件PDQuest 8.0进行图像分析,软件自动给出差异表达蛋白质点的数据信息。切取部分差异明显的蛋白点送香港大学基因研究中心进行鉴定,获得的肽质量指纹图谱,采用Mascot软件在NCBInr数据库中进行搜索,鉴定差异蛋白。部分心肌组织用Western及IP细胞裂解液提取心肌组织蛋白,进行蛋白蛹Ｊ笛椤Ｒ驭?actin为内参,将处理好的蛋白质样品进行SDS-PAGE分离,电泳结束后将蛋白质转移到PVDF膜上,封闭后,孵育一抗和二抗,ECL显色,KODAKImage Station 2000MM成像系统采集图像,获得图像用图像处理软件Image Tool3.0测定并分析条带狄度值以检测FABP的表达水平。三、结果1、定心方及氧化苦参碱对大鼠缺血再灌注心律失常的影响。各组心律失常评分经统计学分析,有显著性差异(x~2=20.614,P=0.000＜0.01)。SH组大鼠偶见室性早搏,未见其它心律失常;I/R组大鼠8例均发生心律失常,多数大鼠冠脉再通1min就开始出现心律失常,以频发室早、室速、室颤等为主,还有少量的房室传导阻滞,其中1例因室颤死亡,VA评分与SH组比较显著增高(P=0.000＜0.01);DXR组8例也均出现心律失常,但以室早和室速为主,VA评分与I/R组相比显著减少(P=0.013＜0.015);OMT组心律失常类型也以室早和室速为主,其中2例仅见偶尔的室性早搏,VA评分与I/R组相比也显著减少(P=0.005＜0.01)。2、定心方及氧化苦参碱对血清ICAM-1表达的影响。各组血清ICAM-1含量经统计学分析有显著性差异(F=4.233,P=0.014＜0.05)。I/R组大鼠血清ICAM-1的含量较SH组显著升高,差异有统计学意义(P=0.007＜0.05);而定心方和氧化苦参碱干预后,血清ICAM-1含量降低,差异有统计学意义(P=0.003、0.049＜0.05),其中定心方与氧化苦参碱相比,定心方有较好的干预作用,但差异无统计学意义(P=0.241＞0.05)。3、再灌注心律失常及定心方和氧化苦参碱干预后大鼠心肌蛋白质表达谱的变化。心肌缺血再灌注后,I/R组蛋白谱与SH组蛋白谱比较,有71个点有明显差异,DXR组有73个蛋白点与I/R组有明显差异,其中I/R组与SH组的差异71个点中,有32个差异点经DXR治疗后,有明显恢复,接近于正常;OMT组与I/R组比较,明显差异的点84个,其中I/R组与SH组的差异71个点中,有30个差异点经OMT治疗后,有明显恢复,接近于正常。经进一步比较,OMT与DXR作用后表达变化一致的点有8个。4、部分差异蛋白点的鉴定。对所获得的部分差异蛋白点进行质谱分析,获得5张肽质量指纹图谱,这些肽质量指纹图谱经Mascot搜索数据库成功鉴定出其中4个蛋白质,它们分别为白蛋白(albumin)、抗增殖蛋白(prohibitin)、心肌脂肪酸结合蛋白(fatty acid binding protein,FABP)、线粒体醛脱氢霉(mitochondrial aldehyde dehydrogenase.mtALDH)。5、FABP蛋白表达水平的验证。为验证质谱鉴定得出的FABP在心肌组织中的表达,我们应用半定量western blot分析检测各组心肌组织中FABP。通过分析结果中目的条带图像发现,各组心肌组织中FABP的表达有显著性差异(F=12.007,P=0.002＜0.05)。I/R组FABP的表达较SH组升高,差异有统计学意义(P=0.000＜0.05),经定心方和氧化苦参碱干预后,FABP的表达明显降低,有显著性差异(P=0.007、0.002＜0.05),与2DE分析结果一致。四、结论1、大鼠缺血再灌注损伤容易诱发心律失常,定心方及氧化苦参碱对再灌注心律失常有明显的保护作用。2、定心方及氧化苦参碱能明显降低再灌注大鼠血清ICAM-1的表达,减轻再灌注心律失常。3、缺血再灌注心律失常及定心方和氧化苦参碱干预均会影响心肌蛋白质的表达谱。4、缺血再灌注心律失常后,心肌能量代谢、自由基清除相关蛋白发生了明显改变。5、蛋白表达水平的验证提示我们建立的双向电泳到质谱鉴定的技术平台相对稳定,在此基础上所做的蛋白质组学研究是可靠可信的。更多还原

【Abstract】 Objective:Along with the wide application of thrombolytic therapy, percutaneous transluminal coronary angioplasty and intracoronary stening to myocardial infarction(MI),myocardial ischemia-reperfusion after acute MI(AMI) becomes more and more popular.According to the experiments and clinic research on AMI,early myocardial ischemia-reperfusion can not only minify infract size and degrade the death rate,but also induce myocardial ischemia-reperfusion injury that causes arrhythmia,myocardial textural change,myocardial stunning and so on. Arrhythrnia is the main clinical manifestation of myocardial ischemia-reperfusion injury,and serious ventricular arrhythmia is the important cause of cardiogenic sudden death,so,it is very significant to study the mechanism of the arrhythmia induced by myocardial ischemia-reperfusion injury and to find the effective medicine to prevent and cure it.Drug treatment is now still the main therapy for arrhythmia though operation and electronic machine have been greatly developed in western medicine.Long-term taking antiarrhythmic drug always bring some adverse reaction,one kind of which the arrhythmia induced by antiarrhythmic drug,have been highly thought of by pharmacist and physician.Inversely,Traditional Chinese Medicine against arrhythmia is characterized by multistrata,multiangular,multitarget and has no significant adverse reaction.So,in this research,by models of the arrhythmia induced by myocardial ischemia-reperfusion injury in rats,we studied the influence of DingXin Recipe and Oxymatrine on arrhythmia,the level of intercellular adhesion molecule-1(ICAM-1) and proteome expression spectra through proteome technique. Through the above-mentioned studies,we expect to reveal the molecular mechanism of arrhythmia induced by myocardial ischemia-reperfusion injury,and the mechanism of DingXin Recipe(DXR) and Oxymatrine(OMT) on arrhythmia,and provide evidence for preventing and curing arrhythmia in clinic induced by myocardial ischemia-reperfusion injury.Methods:32 adult male Wistar rats were randomized into 4 equal groups:Sham operated group(SH),Ischemia-reperfusion group(I/R),DingXin Recipe group(DXR) and Oxymatrine group(OMT).All the rats were fed for 2 days for accommodation. Rats of SH,I/R and OMT groups were drenched with normal sodium,and rats in DXR group were drenched with DXR physic liquor.After successive intragastric administration for 7 days,the rats of I/R,DXR and OMT groups accepted operation, and the rats of SH group also accepted the operation except no ligation and reperfusion.We made models of arrhythmia according to Pharmacological Empirical Methodology.Rats were combined with electrocardiograph and electrocardioscope to observe the occurrence of arrhythmia and made a note of arrhythmia that happened during reperfusion stage and gave a mark of ventricular arrhythmia.After the above-mentioned operation,took blood from abdominal aorta and then prepared serum.Eventually,took out of the heart,washed it in cool normal sodium and separated the left ventricle,then stored it in refrigerator with the temperature of -80℃.Arrhythmia induced by myocardial ischemia-reperfusion injury mainly was ventricular arrhythmias(VA),including premature ventricualr contraction(VE), ventricular tachycardia(VT) and ventricular fibrillation(VF),so we gave a mark of ventricular arrhythmia based on the method of Curtis.We detected ICAM-1 of Serum from abdominal aorta by ABC-ELISA,and observed the effect of DXR and OMT on expression of serum ICAM-1.Total protein was extracted from partial myocardium with lysate,and was quantitated by the method of Bradford.After quantitation,we separated total protein by two-dimensional gel electrophoresis(2DE) technique.IPG3-10L,24cm gel was used in first dimension for level ampholine electrophoresis,and the second dimension was SDS-PAGE vertical electrophoresis.Then Coomassie Brilliant Blue was used to dye the gel when electrophoresis was completed,and images were collected by Umax PowerLook 1100.We analyzed images by PDQuest 8.0 of Bio-Rad company,and software automaticly gave information of differential protein spots.We cut partial differential spots and sent them to Genome Research Centre of the University of Hong Kong.Peptide Mass Fingerprints(PMF) obtained by matrix assisted laser desorption/ionization time-of-flight mass spectrometry/mass spectrometry (MALDI-TOF MS/MS) were searched in NCBInr by Mascot to identify differential protein spots.Total protein was extracted from partial myoeardium with the kit of Western and IP cell lysate for Western blot.We separated total protein by SDS-polyacrylamide gel electrophoresis(SDS-PAGE),transferred total protein to PVDF film after SDS-PAGE, blocked,and incubated antibodies.Then we collected images by KODAK Image Station 2000MM,and analyzed images by Image Tool 3.0 to observe the expression level of fatty acid binding protein(FABP). Results 1.The effect of DXR and OMT on arrhythmia induced by myocardial ischemia-reperfusion injury in rats.By statistics analysis,the score of arrhythmia in different groups had significant difference in total(χ~2=20.614,P=0.000＜0.01).The incidence of premature ventficualr contraction in SH group was low and without other kinds of arrhythmia.In I/R group,arrhythmia occurred in 8 rats,and started to appear when coronary artery was perfused again for 1 minute,mainly including premature ventricualr contraction,ventricular tachycardia and ventricular fibrillation, with a few atrial ventricular blocks,and 1 rat in this group died of ventricular fibrillation.The score of ventricular arrhythmia in I/R group was significantly higher than that in SH group(P=0.000＜0.01).In DXR group,arrhythmia also occurred in 8 rats,mainly premature ventricualr contraction and ventricular tachycardia,and the score of ventricular arrhythmia was lower compared with I/R group(P=0.013＜0.015) In OMT group,arrhythmias were also mainly premature ventricualr contraction and ventricular tachycardia,and there was only premature ventricualr contraction in 2 rats the score of ventricular arrhythmia was also lower compared with I/R group(P= 0.005＜0.01).2.The effect of DXR and OMT on expression of serum ICAM-1.the content of serum ICAM-1 had significant difference by statistics(F=4.233,P=0.014＜0.05).we found that compared with SH group,the content of serum ICAM-1 in I/R group rose obviously,and difference had statistics significance(P=0.007＜0.05);due to the role of DXR and OMT,the content of serum ICAM-1 decreased,and difference had statistics significance(P=0.003,0.049＜0.05),but compared DXR with OMT, difference had no statistics significance(P=0.241＞0.05).3.Changes of protein expression in arrhythmia induced by myocardial ischemia-reperfusion injury and the effect of DXR and OMT.After reperfusion, compared with I/R group,SH group has 71 differential spots,DXR group has 73 differential spots,and in the 71 differential spots of I/R group and SH group,32 differential spots recovered due to the role of DXR;OMT group has 84 differential spots compared with I/R group,and 30 differential spots recovered due to the role of OMT.After advanced comparison,8 differential spots had similar changes due to the role of DXR and OMT.4.Identify partial differential proteins.We identified acquired 5 differential proteins,and obtain five PMFs.These PMFs were respectively identified successfully as 4 proteins after searching database by Mascot:albumin,prohibitin,FABP, mitochondrial aldehyde dehydrogenase(mtALDH).5.Verify the expression of FABP.For verifying the expression of FABP,we detected FABP of myocardium by western blot,and found that the expression of FABP in four groups had significant difference(F=12.007,P=0.002＜0.05);compared with SH group,the expression of FABP in I/R group rose significantly(P= 0.000＜0.05),but due to DXR and OMT,the expression of FABP obviously decreased(P=0.007,0.002＜0.05),which were similar to the results of 2DE.Conclusions 1.Arrhythmia can easily be induced by I/R injury in rats,and it can effectively be prevented by DXR and OMT.2.The expression of serum ICAM-1 was obviously degraded by DXR and OMT, and the occurrence of arrhythmia induced by myocardial ischemia-reperfusion injury was lessened.3.Arrhythmia induced by myocardial ischemia-reperfusion injury,DXR and OMT had effect on maps of protein expression of myocardium.4.After arrhythmia induced by myocardial ischemia-reperfusion injury,proteins related with myocardium energy metabolism and the clearance of free radical had changed obviously.5.Verification of protein expression confirmed that the technology platform of 2DE and mass spectrometry we established was relatively stable,and our study was credible by means of technology platform.更多还原