【作者】 卢军伟；

【导师】 张孟元；

【作者基本信息】 山东大学 ， 麻醉学， 2009， 硕士

【摘要】 研究目的和背景：近40年来,随着体外循环CPB技术和心肌保护方法的发展,在体外循环下进行冠状动脉搭桥术(ONCAB)已成为心肌再血管化标准的常规方法,冠状动脉搭桥术,是让心脏搏出的血从主动脉经过所架的血管桥,流向因引起狭窄或梗阻的冠状动脉远端而到达缺血的心肌,从而改善心肌的缺血、缺氧状态的治疗方式。目前世界上大多数搭桥手术仍在CPB下进行。其优点是心脏停止跳动,术野静止、无血,便于准确完全地进行冠脉搭桥,操作方便安全。大量事实证明其具有良好的早期及远期效果。CPB技术已比较成熟,但对机体来说,仍是一种不正常状态,可带来一系列病理生理紊乱,如炎性反应、血液破坏、血液稀释、颗粒和异物栓子形成、缺血再灌注损伤,可能加重严重肺、脑、肾患者的病情。近十年来,随着医学发展和技术的进步,开展了通过心脏不停跳和不需要体外循环的方法来完成搭桥手术,即非体外循环心脏跳动下搭桥手术(OPCAB)。这也是心脏稳定装置的不断改进与心脏外科手术完美结合的结果。它使心脏需要搭桥的一小部分保持极小的运动状态,而整个心脏的绝大部分在正常跳动和持续工作为全身供血,减少了心肌再灌注损伤,成功的减少了手术并发症和促进早期康复。非体外循环心脏跳动下搭桥手术拓宽了冠脉搭桥手术的适应证。对于高危病人,尤其是伴有肺、肾、神经系统以及严重左心功能不全的病人更适合于非体外循环心脏跳动下搭桥。这种方法使手术后的恢复过程更平稳顺利。术后发生重要脏器功能衰竭的发生率减少。如今,非停跳冠脉搭桥(OPCABG)虽有取代停跳冠脉搭桥(CABG)的趋势,但其本身始终也存在缺血再灌注损伤的问题。只有充分认识和解决OPCABG中缺血再灌注损伤这一问题,心脏才会得到强有力的保护。Toll样受体是天然免疫系统识别病原微生物的主要受体,在天然免疫反应中具有重要作用。Toll样受体4(TLR4)不仅是介导脂多糖信号跨膜转导的主要受体,而且也参与了缺血性损伤等非病原微生物性炎症反应。有关TLR4及其介导的信号转导在冠心病中的作用日益引起人们的关注。深入研究TLR4在冠心病中的作用,有可能为进一步揭示冠心病的发病机制并为寻求有效的防治措施提供重要的理论基础。材料与方法：本研究项目首先以SD大鼠建立OPCABG模型,然后获取心肌组织经电镜观察其超微结构变化,同时采用分子生物学方法分析心肌细胞中TLR4与mRNA,HSP60在不同时间段活性表达的变化,以期明确大鼠OPCABG中上述观察指标之间的关系。1动物实验：以SD大鼠为实验对象,由山东大学实验动物研究所提供,其TLR4基因存在点突变,对内毒素天然耐受。30只大鼠,16-18周龄,体重250-320g。共分2个小组,假手术组(Sham组)、缺血再灌注组(IR组)。缺血再灌注组大鼠被钳夹冠脉主干,3分钟后撤去钳夹,以此模仿冠脉搭桥后造成的急性缺血再灌注损伤。每组再分5个时间点,分别为冠脉主干钳夹后0、1、6、12、24h,在每个时间点上处死3只大鼠。在上述时间点,给以腹腔内注射过量的戊巴比妥钠(80mgPkg)处死大鼠,快速低温剪取心肌组织。2心脏TLR4mRNA表达按说明书操作进行PCR反应。3心脏组织热休克蛋白60(HSP60)含量检测应用HSP-AELISA试剂盒检测,具体操作按说明书进行.4心脏组织电镜下观察实验结束时留取大鼠心肌组织,电镜下观察超微结构变化。5统计学处理目的基因电泳条带密度值以灰度@面积、参照物的值表示。所有数据用SPSS软件统计处理,数据均以x±s表示,统计学处理采用t-test。结果：1.应用酶联免疫吸附测定(ELISA)检测HSP60水平并进行对比分析。结果在假手术组和缺血再灌注组HSP60水平间差别有显著性意义(P＜0.01)；缺血再灌注组HSP60水平明显高于假手术组。2. Sham组心肌组织形态及超微结构改变不明显；IR组心肌损伤较重,缺血心肌恢复血液灌注8h内,其病理学变化未见显著改善。3. Sham组与IR组TLR4蛋白都有阳性表达,IR组TLR4表达增加,且以再灌注1h最明显(19.62±3.84,P(0.01)。4.与Sham组比较,IR组心肌TLR4mRNA表达水平均出现不同程度上调,以再灌注1h达到峰值[(4.03±0.85)×10^-2,P＜0.01],而Sham组各时间点未见明显改变。5.心肌TLR4mRNA表达与HSP呈正相关(r=0.728,P(0.01)。结论：1.心肌Toll样受体4与mRNA表达水平呈正相关表达,TLR4在心肌早期心肌缺血再灌注后的上调和活化表明Toll样受体4在心肌缺血再灌注中可能发挥重要作用。2.热休克蛋白60阳性染色,观察缺血再灌注组,Toll样受体4与蛋白的水平明显增加,而且高峰在1小时后灌注。此外,在缺血再灌注组,Toll样受体4与mRNA水平也上调了。3.TLR4水平与mRNA,HSP,表达积极。心肌Toll样受体4表达在早期心肌缺血再灌注时升高。Toll样受体4激活可能在心肌组织缺血再灌注中发挥重要作用,增加mRNA与HSP的表达。4.心肌缺血再灌注早期,心肌TLR4表达迅速上调,TLR4的激活可能通过促进TNF-Q等炎性因子的产生分泌增多来介导心肌缺血再灌注损伤。5.缺血再灌注组病TLR4 mRNA表达、HSP表达均显著高于假手术组。TLR4可能通过促使炎性因子产生分泌增多来介导脑缺血炎性损伤。更多还原

【Abstract】 Background and Objective:Over the past 20 years, China’s year-on-year increase in the incidence of coronary heart disease, faster growth in males. The incidence of coronary heart disease from 1980 million to the 27/10 of 78/10 million in 2005.1980 to 2005 the incidence for men increased by 67 percent, the Ministry of Health data reported that the city of coronary heart disease deaths 71.3/10 million in rural areas of 31.6/10 million. Treatment to improve the level of coronary heart disease has become one of the important tasks of medical workers.Nearly 40 years, with the CPB pump technology and the development of methods of myocardial protection in off-pump coronary artery bypass grafting (ONCAB) myocardial revascularization has become the standard of the conventional methods, coronary artery bypass grafting, cardiac pacing is a blood from the aorta through the vascular frame bridge, caused by the flow due to stenosis or obstruction of the coronary artery to reach the remote and ischemic myocardium, thereby improving myocardial ischemia, hypoxia. Pathophysiology of coronary heart disease is characterized by:when the coronary blood supply is inadequate relative or absolute, myocardial metabolic shift from aerobic metabolism of anaerobic metabolism, the supply of myocardial energy (ATP) quickly reduced the gradual cessation of cardiac contractility. This change in acute coronary occlusion in a few minutes, but even the most severe myocardial ischemia in 15-18 minutes, the survival of myocardial cells at this time if the blood re-perfusion, ischemic injury to stop and re-perfusion After 1-4 days, there was no necrosis of myocardial cells, such as the extension of ischemic time, it will not be able to reperfusion to prevent the death of myocardial cells, the longer the ischemic time, irreversible damage of the cells more ischemic 40-60 minutes After the central area of severe ischemia in the majority of dead cells, moderate ischemia of the middle and epicardial cells can survive a longer period of time, six hours after coronary occlusion, the cells most affected by the disposal of dead.At present, most of the world’s bypass surgery is still under CPB. The advantage of cardiac arrest, surgical field static, non-blood, completely and accurately to facilitate coronary artery bypass grafting, easy to operate security. A large number of facts to prove their good early and long-term effect. CPB technology is more mature, but the body is still an abnormal state can be brought about by a series of pathophysiological disorders such as inflammatory response, blood damage, blood dilution, particles and the formation of foreign body emboli, ischemia reperfusion injury may increase the serious lung, brain, kidney patients.Over the past decade, with medical advances and technological progress, carried out through the beating heart and the method does not require cardiopulmonary bypass to complete the bypass surgery, that is, off-pump beating heart bypass surgery (OPCAB). This is the heart of the continuous improvement of the stability of devices with the perfect combination of cardiac surgery outcome. It makes the need to bypass the heart of a small part of the movement to maintain a very small state, but the vast majority of the entire heart beating in the normal body work and sustainable blood supply, a reduction of myocardial reperfusion injury, a successful surgery to reduce complications and promote early rehabilitation. Off-pump beating heart bypass surgery broadens the indications for coronary artery bypass surgery. For high-risk patients, especially those with lung, kidney, nervous system, as well as severe left ventricular dysfunction in patients more suitable for off-pump beating heart bypass. So that the complexity of the surgery was becoming simplified, but the surgeon to anesthesia and higher technical requirements. This approach to make the recovery process after surgery is more smooth. Less use of positive inotropic drugs, from the ventilator earlier and shorten ICU length of stay in hospital wards and to reduce the possibility of secondary thoracotomy to stop bleeding. Less surgical blood transfusion. Important post-operative organ failure to reduce the incidence. Reduces the nervous system, kidneys, lungs and other complications, exclude a number of cardiac arrest and cardiopulmonary bypass-related complications and reduce the cost of treatment.Today, the non-pump coronary artery bypass grafting (OPCABG) to replace the pump coronary artery bypass grafting despite (CABG) the trend, but there has always been its own ischemia-reperfusion injury problems. Only by fully understanding and resolving OPCABG in ischemia-reperfusion injury in this issue, the heart will be a strong protection.Material and methodsFirst of all, this research project to establish OPCABG model of SD rats, and then access to myocardial tissue by electron microscopy the ultrastructure changes in the methods of analysis of the use of molecular biology of cardiac myocytes TLR4 and mRN A, HSP60 activity in different time periods of expression changes rats with a view to a clear indicator of OPCABG in the above-mentioned relationship between the observed and the corresponding morphological changes.1 animal experiments:to SD rats for the experiment, from Shandong University, Institute of Laboratory Animal, the TLR4 gene point mutation, natural tolerance to endotoxin.30 female rats,16 to 18 weeks of age, body weight 250-320g, reared in a clean environment, free drinking water,12h light. Is divided into two groups of mice were coronary trunk clamp,30 minutes back to the clamp in order to mimic a result of coronary artery bypass grafting after acute ischemia-reperfusion injury. Further divided each of five time points, respectively, after coronary trunk clamp 0,1,6,12,24 h, at each time point three mice were killed. In the above-mentioned point in time, given intraperitoneal injection of pentobarbital sodium overdose (80mgPkg) rats were killed, and rapid low-temperature剪取myocardial tissue for RNA extraction, followed by TLR4 and mRNA, HSP60, the expression of detection, as well as optical and electron microscopy observation of histological changes.2 expression in the heart of TLR4mRNAThat heart tissue from 100-120mg, adding RNA extraction reagent 1mL, electric glass into homogenate homogenate mechanism, operated by manual extraction of total RNA "and the use of reverse transcriptase Oligod (T) 15 primer reverse transcription of total RNA to cDNA, for PCR reaction.3 heart tissue heat shock protein 60 (HSP60) content detectionApplication of HSP-AELISA detection kit, the specific operation carried out according to specification.4 electron microscope observation of heart tissueCollecting the end of the experiment when the rat cardiac tissue, malondialdehyde saved, and then fixed, embedded, slicing, staining, electron microscopy ultrastructural changes.5 statisticalGene density of electrophoretic bands @ a gray area, said reference value. All data processing with SPSS statistical software, data are x±s, said the use of statistical t-test.Result1. Application of enzyme-linked immunosorbent assay (ELISA) detecting the level of HSP60 and analyzed. Results in the sham operation group and ischemia-reperfusion group the differences between the level of HSP60 was significant (P<0.01); ischemia-reperfusion group the level of HSP60 was significantly higher than sham-operated group.2.Sham group myocardial tissue was not obvious change in morphology and ultrastructure; IR group myocardial injury heavier, the restoration of blood perfusion in ischemic myocardium 8h, its pathological changes not significantly improved.3. Sham group and the IR group have positive expression of TLR4 protein, IR group increased TLR4 expression, and to the most obvious 1h of reperfusion (19.62±3.84, P<0.01).4. With the Sham group, IR group myocardial expression TLR4mRNA with varying degrees of water increases the average to peak 1h reperfusion [(4.03±0.85)×10 (?) -2, P<0.01], and the Sham group at different time points not significant change. 5. TLR4mRNA myocardial HSP expression was positively correlated (r= 0.728, P＜0.01).Conclusion1. Myocardium Toll-like receptor 4 expression levels and mRNA expression was positively correlated, TLR4 in early myocardial reperfusion after myocardial ischemia and activation showed that the increase in Toll-like receptor 4 in myocardial ischemia-reperfusion may play an important role.2. Heat shock protein 60-positive staining was observed in ischemia-reperfusion group, Toll-like receptor 4 and a marked increase in protein levels, and peak at 1 hour after reperfusion. In addition, ischemia-reperfusion group, Toll-like receptor 4 mRNA levels are up.3. TLR4 level of mRNA, HSP, the positive expression. Myocardial Toll-like receptor 4 expression in early myocardial ischemia-reperfusion increased. Toll-like receptor 4 activation may be in the myocardial tissue ischemia and reperfusion play an important role in increasing the expression of mRNA and HSP.4. Early myocardial ischemia-reperfusion, the rapid increase in cardiac TLR4 expression, TLR4 activation may be through the promotion of TNF-a and other inflammatory factors to produce an increase in the secretion-mediated myocardial ischemia-reperfusion injury.5. Ischemia-reperfusion group TLR4 mRNA expression of disease, HSP expression were significantly higher than sham-operated group. TLR4 inflammatory factors may have led to increased secretion of cerebral ischemia-mediated inflammatory injury.更多还原