【作者】 张铮；

【导师】 王海昌； 曹丰；

【作者基本信息】 第四军医大学 ， 内科学， 2010， 硕士

【摘要】 实验背景及目的:心肌缺血再灌注损伤(myocardium ischemia/reperfusion injury, MIRI)是冠心病心肌梗死溶栓和介入治疗的重要病理生理过程。失控的炎症反应是MIRI损伤的重要特征。心肌微血管内皮细胞(Cardiac microvascular endothelial cells, CMEC)是心脏微循环的重要组成部分,也是MIRI过程中最先波及且最先受损的部位,因此在MIRI损伤机制中发挥着重要作用。Toll样受体4(TLR-4)为体内模式识别受体Toll样受体家族的主要成员,由于能够识别多种内源性和外源性配体,不仅可介导感染性炎症反应,在非感染性炎症反应中也发挥着重要作用。近年来TLR-4在MIRI中的作用也受到越来越多的关注。最新的研究表明:TLR-4基因缺陷小鼠(C3H /HeJ小鼠)与野生型小鼠(C3H/HeN小鼠)相比MIRI后的心肌梗死面积明显减少。进一步研究也证实,给予TLR-4特异性抑制剂eritoran阻断TLR-4信号通路能够对小鼠MIRI起到保护作用。因此,我们推测TLR-4信号通路可能参与了MIRI的炎症反应。但是,目前的研究主要集中于TLR-4信号通路在心脏组织水平以及心肌细胞I/R损伤中的作用,而在CMEC I/R损伤中的作用尚无报道。因此,本研究试图通过以下方法:1、体外分离培养CMEC;2、建立缺氧/复氧(H/R)模型,模拟I/R损伤;3、观察H/R损伤中TLR-4信号通路的变化;4、观察应用TLR-4中和性抗体阻断TLR-4信号通路是否对CMEC I/R损伤具有保护作用,探讨TLR-4信号通路在CMEC MIRI损伤中的作用。实验方法第一部分:CMEC的分离、培养及缺氧复氧模型的建立无菌条件下取出大鼠心脏,利用化学消化法成功分离CMEC。用含15%胎牛血清(FCS)的DMEM培养液维持培养。取2-3代的CMEC缺氧6h后,分别复氧2 h、12 h及24 h,采用四甲基偶氮唑蓝(MTT)比色法检测内皮细胞的增殖能力;Hochest染色检测细胞凋亡;硝酸还原法测定细胞培养液中一氧化氮(NO)分泌量;细胞划痕实验检测细胞迁移能力。第二部分:TLR-4信号通路在CMEC缺氧复氧损伤中的变化取体外培养2-3代的CMEC缺氧6 h后,分别复氧2 h、12 h及24 h。收集细胞培养液,并裂解细胞,获得CMEC蛋白。用Western blot方法检测细胞TLR-4及NF-κB蛋白表达水平。用ELISA试剂盒检测培养液中炎性因子IL-6、TNF-α分泌水平。第三部分:抑制TLR-4信号通路对CMEC缺氧复氧损伤的保护作用取体外培养2-3代的CMEC,给予TLR-4中和性抗体MTS510(10ug/ml) 37℃孵育2h后给予H/R处理。缺氧6 h后复氧2h,再次用四甲基偶氮唑蓝(MTT)比色法检测内皮细胞的增殖能力;用Hochest染色检测细胞凋亡;硝酸还原法测定细胞培养液中NO分泌量;利用细胞划痕实验检测细胞迁移能力。实验结果1.体外成功分离、培养大鼠CMEC。2.与正常对照组相比,经过H/R损伤后,各组CMEC的增殖均明显受到抑制(P<0.05),细胞凋亡率增加(P<0.05);CMEC的NO分泌能力及迁移能力均显著下降(P<0.05)。3.与正常对照组相比,TLR-4蛋白水平在复氧后2 h及12 h明显升高(P<0.05),24 h基本恢复正常;NF-κB蛋白水平在复氧2 h、24 h显著高于对照组(P<0.05);而CMEC在复氧2 h、12 h及24 h后分泌的IL-6和TNF-α水平均高于对照组(P<0.05)。4.通过特异性中和性抗体阻断TLR-4信号通路,CMEC在缺氧6h复氧2h后,与单纯H/R处理组相比,增殖能力显著增加(P<0.05),细胞凋亡率下降(P<0.05);细胞迁移能力提高(P<0.05);NO分泌能力以及eNOS表达水平显著增加(P<0.05)。结论1. H/R损伤可以导致CMEC增殖能力下降,凋亡增加,并导致细胞迁移能力、NO分泌下降等功能障碍。2.在H/R早期,TLR-4/NF-κB信号通路激活,导致下游炎性因子IL-6、TNF-α分泌的增加,从而参与CMEC的炎症反应。3.通过特异性中和抗体抑制TLR-4/NF-κB信号通路,可以减少H/R诱导的CMEC凋亡及功能障碍,对CMEC的H/R损伤具有保护作用。更多还原

【Abstract】 BackgroundMyocardium ischemia/reperfusion injury remains the major cause of cardiac dysfunction in human cardiovascular pathophysiology since it occurs in a wide variety of ischemic cardiovascular disorders such as myocardial ischemia and stroke. Microcirculation dysfunction is an important etiological component of MIRI. Accumulating evidence suggests that I/R injury can induce ECs malfunction which is the most important reason of microcirculation dysfunction. Moreover, in the very early stages of reperfusion, ECs apoptosis is firstly observed, which precede myocyte cell apoptosis. Some soluble inflammation mediators from damaged ECs are shown to induce myocyte apoptosis. However, the mechanism of ECs dysfunction induced by MIRI is still not well investigated.Toll-like receptor (TLR) family consists of highly conserved pattern recognition receptors. Among the 10 TLRs identified in human, TLR-4 is the most extensively investigated since its recognition of lipopolysaccharide (LPS). However, evidences show that the ligands of TLR-4 are not only LPS, but also some specific endogenous molecules, such as fibronectin, fibrin, extracellular matrix fragments, heat-shock proteins 60 (HSP-60), HSP-70 and so on. TLR-4, initially found in monocytes, has been shown to be expressed in many tissues, including cardiomyocytes and ECs. Recently, the myocardial MIRI has been viewed as an innate immune response which is mediated in part through TLR-4 signal pathway. The TLR-4 deficient mice had reduced myocardial MIRI injury. Moreover, inhibition of TLR-4 signal with eritoran also attenuated cardiac dysfunction induced by MIRI injury. Previous clinical studies indicate that TLR-4 activation plays an important role in pathology process of atherosclerosis and heart failure.Although several studies have shown that TLR-4 takes part in MIRI of heart, most current studies focused on the relationship between TLR-4 and global heart dysfunction or cadiomyocyte apoptosis. The effect of TLR-4 on CMEC in MIRI is not clear so far. We hypothesized that TLR-4 signaling modulates CMEC dysfunction post MIRI.MethodsPart I: CMEC were isolated from adult male Sprague-Dawley rat hearts. Cultured CMEC, after being replaced with hypoxic buffer, were exposed to hypoxia in anaerobic system at 37℃for 6h. For reoxygenation, CMEC, after hypoxia treatment, were removed from the anaerobic chamber and then maintained in a regular incubator for 2h, 12h and 24h respectively. The proliferation of CMEC was assessed by MTT assay, the apoptosis of CMEC was detected by Hocheset staining, the migration of CMEC was detected by cell scratch wound healing assay and the level of nitric oxide (NO) was detected by an NO assay kit according to the manufacturer’s protocol.Part II: After CMEC were treated with hypoxia for 6h followed by reoxygenation 2h, 12h, 24h respectively, the expressions of TLR-4, Nuclear Factor-kappa B (NF-κB) were analyzed by Western blotting. And the levels of interleukin-6 (IL-6), Tumor necrosis factor-α(TNF-α) were detected by ELISA kits.Part III: CMEC were divided into four groups: (1) control group, (2) H/R group, (3) TLR-4 blocking group, (4) TLR-4 blocking and H/R group. The TLR-4 signal of CMEC was blocked by administration with the neutralizing antibody MST510 (10ug/ml). The H/R treatment was performed as hypoxia 6h followed with reoxygenation 2h. Like the methods used in Part I, the proliferations of CMEC in all groups were assessed by MTT assay, the apoptosis of CMEC was detected by Hocheset staining, the migrations of CMEC were detected by cell scratch wound healing assay, the levels of NO, IL-6, TNF-αwere detected by kits.Results1. CMEC were isolated and cultured in vitro successfully.2. Proliferation and migration of CMEC were impaired after H/R injury (P<0.05 vs. control). Moreover, H/R injury increased the apoptosis of CMEC (P<0.05 vs. control), and decreased the level of NO secretion (P <0.05 vs. control).3. Compared with control, H/R injury increased TLR-4 expression after 2 h or 12 h reoxygenation (P<0.05 vs. control). The level of NF-κB increased after 2 h and 24 h reoxygenation (P<0.05 vs. control). H/R injury also enhanced IL-6 and TNF-αsecretion as compared with control group (P<0.05 vs. control).4. Blocking TLR-4 signal pathway by administration of the neutralizing antibody MTS510 prior to H/R treatment attenuated the proliferation and migration of CMEC after H/R injury (P<0.05 vs. H/R). Moreover, TLR-4 inhibition decreased the apoptosis of CMEC after H/R injury (P<0.05 vs. H/R), and increased the NO level (P<0.05 vs. H/R).Conclusions1. H/R injury can increase the apoptosis of CMEC, and impair the proliferation. Moreover, H/R also induces CMEC dysfunction of migration and secretion of NO.2. In the early state of H/R injury, TLR-4/NF-κB signal pathway in CMEC is activated and the activation of TLR-4/NF-κB pathway enhances the secretions of IL-6 and TNF–α, which may participate in the injury and dysfunction of CMEC caused by H/R.3. Blocking TLR-4 signal decreases the apoptosis of CMEC and attenuates dysfunction caused by H/R, suggesting a possible mechanism by which H/R injury lead to CMEC dysfunction.更多还原