【作者】 侯丽芳；

【导师】 盘强文；

【作者基本信息】 泸州医学院 ， 生理学， 2010， 硕士

【摘要】 目的：研究无创肢体缺血预处理对兔缺血性室性心律失常的影响及其作用机制,为其临床应用提供实验性理论依据。方法：日本大耳白兔27只,体重(1.8±0.4)kg,雌雄不拘,随机分至单纯缺血对照组(Ctr,n=8)、无创肢体缺血预处理组(LIP,n=7)、LIP+格列本脲组(LG,n=6)和LIP+SB203580组(LSB,n=6)。动物麻醉后,经右颈外静脉插入电极至右心室记录心内膜MAP；针形电极插入四肢皮下记录标准Ⅱ导联ECG；经胸骨左缘第4或第5肋间隙开1-2mm2小孔,插入接触电极至心室外心包壁层稍加压记录心外膜MAP。Ctr组不作特殊处理,待ECG和心内、外膜MAP图形稳定后,静脉注射垂体后叶素(2U/kg)造成心肌缺血背景,再静脉注射肾上腺素(0.2ml/kg)诱发心律失常；同步记录ECG及心内、外膜MAP,观察心内、外膜MAP变化及与心律失常间的关系；LIP组：用充气止血带结扎兔双后肢使之缺血5min,松开使之再灌注5min,重复4次,余同Ctr组；LG组：先静脉注射格列本脲(0.16mg/kg),30min后行与LIP组相同的处理；LSB组：先静脉注射SB203580(0.1mg/kg),30min后行与LIP组相同的处理。用RM6280生物信号采集处理系统动态观察ECG和心内外膜MAP的变化。实验结束后,空气栓塞处死动物,摘取心脏,部分用4%多聚甲醛固定做形态学观察,部分存于-70℃冰箱待用。结果：①缺血前后,Ctr组心内膜APD90分别为(130.66±13.36)ms和(135.23±10.24)ms,心外膜APD90分别为(132.56±16.27)ms和(141.90±16.37)ms；LIP组心内膜APD90分别为(122.47±13.94)ms和(130.26±17.12)ms,心外膜APD90分别为(124.16±9.72)ms和(148.02±12.78)ms；LG组心内膜APD90分别为(127.19±36.78)ms和(134.72±46.01)ms,心外膜APD90分别为(146.20±16.21)ms和(160.78±22.73)ms；LSB组心内膜APD90分别为(100.88±13.36)ms和(104.56±28.46)ms,心外膜APD90分别为(105.77±18.59)ms和(104.10±20.69)ms。缺血后各组均出现室性心律失常,主要为室性早搏二联律和三联律。Ctr组有3例出现室性心动过速,未出现心室颤动,LIP、LG及LSB组均未出现室性心动过速及心室颤动。心律失常持续时间(s)分别为(231.54±15.78),(167.61±16.13),(159.38±29.56)和(209.83±26.39)。②心肌组织中SOD活力(U/mgprot)分别为(263.78±61.51),(322.63±50.46),(280.56±37.01)和(314.52±44.72)；MDA含量(nmol/mgprot)分别为(14.01±6.23),(5.34±2.17),(6.25±2.57)和(5.94±2.13)；NO含量(μmol/gprot)分别为(30.85±10.13),(242.17±48.84),(68.18±31.26)和(65.18±22.55)。③缺血后,各组心内膜APD90均延长,LIP及LSB组心内膜APD90短于Ctr组(P<0.05)；缺血后Ctr、LIP及LG组心外膜APD90延长而LSB组APD90缩短,LSB组心外膜APD90短于Ctr及LIP组(P<0.05)。LIP及LG组心律失常持续时间短于Ctr组(P<0.05)。LIP组SOD活力高于Ctr组(P<0.05),LG及LSB组SOD活力与Ctr及LIP组相比无差异(P>0.05);LIP、LG及LSB组MDA含量均低于Ctr组(P<0.01),LG及LSB组MDA含量与LIP组相比无差异(P>0.05)；LIP组NO含量高于Ctr组(P<0.01),LG及LSB组NO含量低于LIP组(P<0.01),LG及LSB组NO含量与Ctr组相比无差异(P>0.05)。④HSP70 mRNA的相对表达量分别为(3.04±0.20),(3.14±0.20),(2.65±0.18)和(2.61±0.31),LIP组HSP70 mRNA相对表达量与Ctr组相比无差异(P>0.05),而LG及LSB组HSP70 mRNA的相对表达量均低于Ctr及LIP组(P<0.01)；COX-2 mRNA的相对表达量分别为(1.36±0.16),(1.61±0.22),(1.47±0.19)和(1.14±0.09),LIP组COX-2 mRNA相对表达量高于Ctr组(P<0.05),LSB组COX-2 mRNA相对表达量低于Ctr及LIP组(P<0.05),LG组COX-2 mRNA相对表达量与Ctr及LIP组相比均无差异(P>0.05)；iNOS mRNA的相对表达量分别为(1.32±0.08),(1.75±0.12),(1.40±0.14)和(1.34±0.07),LIP组iNOS mRNA的相对表达量高于Ctr组(P<0.01),LG及LSB组iNOS mRNA的相对表达量低于LIP组(P<0.01)；TNFa mRNA的相对表达量分别为(0.57±0.03),(0.38±0.03),(0.55±0.06)和(0.54±0.04),LIP组TNFa mRNA的相对表达量低于Ctr组(P<0.01),LG及LSB组TNFa mRNA的相对表达量高于LIP组(P<0.01)；NF-κB mRNA的相对表达量分别为(0.62±0.05),(0.51±0.03),(0.59±0.04)和(0.60±0.04),LIP组NF-κB mRNA的相对表达量低于Ctr组(P<0.01),LG及LSB组NF-κB mRNA的相对表达量高于LIP组(P<0.01)。⑤p-p38MAPK蛋白阳性表达量在Ctr组为(0.08±0.02),LIP组为(0.15±0.03),LG组为(0.12±0.02),LIP及LG组p-p38MAPK蛋白表达均高于Ctr组(P<0.05),而p-p38MAPK蛋白在LSB组中的阳性表达量为(0.05±0.02),与Ctr、LIP及LG组相比均减少(P<0.05)。结论：1.LIP具有减轻心肌氧化损伤和改善心肌供血,进而增强兔缺血心肌电稳定性的作用；2.p38MAPK是LIP增强缺血心肌电稳定性的重要途径,KATP则在抗氧化和改善心肌供血等方面发挥了作用；3. LIP对I/R心肌起保护作用可能的机制和信号转导通路是,首先经“触发物质”启动心肌保护,然后通过“中介物质”启动信号转导,激活下游丝裂素活化蛋白激酶(MAPK)系统,进而引起信号的传递和放大,并活化核转录因子及产生“效应子”最终发挥心肌保护作用。更多还原

【Abstract】 Abstract:Objective:To study on effects and mechanisms of the non-invasive limb ischemic preconditioning on ischemic ventricular arrhythmias in rabbits thus provides a practically theoretical basis for its clinical trial.Methods:Twenty-seven rabbits had been randomly divided into the control group(Ctr, n=8),non-invasive limb ischemic precondi-tioning group(LIP, n=7), LIP+ glibenclamide group(LG, n=6) and LIP+ SB203580 group(LSB,n=6).After anesthesia, the right external jugular vein was dissected and cannulated to the right ventricle to record the endocardium MAP. Needle electrodes were punctured in four limbs of the rabbit to record electrocardiogram (ECG).The contact electrode was inserted from the 4th or 5th ribs beside the left sternal to record the epi-cardium monophasic action potential(MAP).Ctr group does not been given any special treatment, but the vasopressin (2U/kg) was injected intravenously to produce myocardial ischemia background, then epine-phrine (0.2ml/kg) was injected to induce arrhythmia, when the records of ECG, endocardium and epicardium MAP were stabilized.The ECG, endo-cardium and epicardium MAP were simultaneously recorded, and the changes of endocardium & epicardium MAP and the relationship to arr- hythmia were observed.LIP group:pneumatic tourniquet was used to occlude the blood flow of rabbits’two hind limbs make them ischemia for 5 minutes,then loosened to reperfusion for 5 minutes,and repeated it for four times.Another methods and contents of observation were same as the Ctr group.LG group:Firstly, glibenclamide(0.16mg/kg) was intra-venous injection,30 minutes later, to carry out the same treatments as the LIP group.LSB group:After the SB203580 (0.1mg/kg) was intravenous injection for 30 minutes,to carry out the same treatment as the LIP group. ECG, the changes of endocardium & epicardium MAP, the types,inci-dence and duration of arrhythmia were simultaneously recorded.At the end of the experiment, air embolism was used to kill the animals, then remove the heart, and some parts were fixed with 4% paraformaldehyde for morphological observation, the other parts were stored in-70℃refri-gerator for another detections.Results:①Before and after ischemia, endocardium APD90 were(130.66±13.36)ms and(135.23±10.24)ms, epicardium APD90 were(132.56±16.27)ms and(141.90±16.37)ms in the Ctr group.Endocardium APD90 were(122.47±13.94)ms and(130.26±17.12)ms, epicardium APD90 were(124.16±9.72)ms and(148.02±12.78)ms in the LIP group.Endocardium APD90 were(127.19±36.78)ms and (134.72±46.01)ms, epicardium APD90 were (146.20±16.21)ms and (160.78±22.73)ms in the LG group.Endocardium APD90 were(100.88±13.36)ms and(104.56±28.46)ms,epicardium APD90 were(105.77± 18.59)ms and(104.10±20.69)ms in the LSB group.After ischemia, all rabbits presented arrhythmia, mainly represented as frequent ventricular premature contraction of bigeminy or trigeminy. There were 3 cases of ventricular tachycardia and no ventricular fibrillation in the Ctr group. There was no ventricular tachycardia and ventricular fibrillation in the LIP, LG and LSB group.The duration of arrhythmia(s)was (231.54±15.78),(167.61±16.13),(159.38±29.56) and (209.83±26.39) respec-tively.②The heart tissue detection of Ctr, LIP, LG & LSB group respectively indicated,SOD activity (U/mgprot) was (263.78±61.51), (322.63±50.46),(280.56±37.01)and(314.52±44.72).MDA content (nmol/mgprot) was(14.01±6.23),(5.34±2.17),(6.25±2.57) and (5.94±2.13).NO content(μmol/gprot) was (30.85±10.13),(242.17±48.84), (68.18±31.26)and(65.18±22.55).③After ischemia,endocardium APD90 was extended in each group,but endocardium APD90 of LIP and LSB group were shorter than Ctr group(P<0.05).Epicardium APD90 of Ctr group, LIP and LG group were extended while LSB group was shortening, and epicardium APD90 of the LSB group was shorter than Ctr and LIP group(P<0.05).The duration of arrhythmia of the LIP and LG group was shorter than the Ctr group(P<0.05).SOD activity of LIP group was higher than Ctr group(P<0.05).SOD activity of LG and LSB group compared with Ctr and LIP group no significant difference(P>0.05).The MDA levels of LIP, LG and LSB group were lower than Ctr group (P<0.01),and the MDA content of LG and LSB group compared with LIP group no significant difference(P>0.05).The NO content of LIP group was higher than Ctr group(P<0.01),and the NO content of LG and LSB group were lower than LIP group(P<0.01).④The relative expres-sion of HSP70 mRNA was(3.04±0.20),(3.14±0.20),(2.65±0.18)and (2.61±0.31)respectively. The relative expression of LIP group HSP70 mRNA compared with the Ctr group no significant difference(P>0.05), while the relative expression of LG and LSB group HSP70 mRNA were lower than Ctr and LIP group(P<0.01).The relative expression of COX-2 mRNA was(1.36±0.16),(1.61±0.22),(1.47±0.19)and(1.14±0.09) respectively. The relative expression of LIP group COX-2 mRNA was higher than Ctr group(P<0.05),and the relative expression of LSB group COX-2 mRNA was lower than Ctr and LIP group(P<0.05),while the relative expression of LG group COX-2 mRNA compared with Ctr and LIP group no significant difference(P>0.05).The relative expres-sion of iNOS mRNA was(1.32±0.08),(1.75±0.12),(1.40±0.14)and(1.34±0.07) respectively. The relative expression of LIP group iNOS mRNA was higher than Ctr group(P<0.01),and the relative expression of LG and LSB group iNOS mRNA were lower than LIP group(P<0.01).The rela-tive expression of TNFa mRNA was(0.57±0.03),(0.38±0.03),(0.55 +0.06) and (0.54±0.04)respectively. The relative expression of LIP group TNFa mRNA was lower than Ctr group(P<0.01),and the relative expression of LG and LSB group TNFαmRNA were higher than LIP group(P<0.01).The relative expression of NF-κB mRNA was (0.62±0.05),(0.51±0.03),(0.59±0.04)and (0.60±0.04)respectively. The relative expression of LIP group NF-κB mRNA was lower than Ctr group(P<0.01),and the relative expression of LG and LSB group NF-κB mRNA were higher than LIP group(P<0.01).⑤The protein expression of p-p38MAPK was (0.08±0.02)in the Ctr group,(0.15±0.03)in the LIP group, (0.12±0.02)in the LG group, The protein expression of p-p38MAPK in the LIP and LG group were higher than Ctr group (P<0.05).While it was (0.05±0.02)in the LSB group,and lower than the Ctr, LIP and LG group(P<0.05).Conclusion:1.LIP can reduce the injury of ischemic myocardial oxidative and improve myocardial blood supply, thus enhance electrical stability of ischemia myocardium.2.p38MAPK plays a very important role on LIP increasing cardiac electrophysio-logical stability, but KATP may participate only in antioxidation and to improve the blood supply of myocardium.3.The possible mechanisms and pathways of signal transduction of LIP protecting the I/R myocar-dium as follow. At first, LIP produce the "triggering substances" to switch on myocardial protection, then through the "intermediary" to start signal transduction, after that, mitogen-activated protein kinase system was activated to induce the signal transmission and amplification, and the activation of nuclear transcription factors,then the production of "effect- or" educe the myocardial protection effects.更多还原