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C5a对血管内皮细胞表达血栓调节蛋白的影响
Effect of C5a with the Thrombomodulin Expression on Endothelial Cells in Vitro
【作者】 房巍；
【导师】 郭振辉；
【作者基本信息】 广州医学院 ， 急诊医学， 2009， 硕士
【摘要】 脓毒症导致多脏器功能障碍综合征(MODS)的发病机理主要与失控的宿主防御反应导致炎症、内皮损伤、凝血亢进、纤溶减弱等有关。血管内皮细胞(VEC)是血管内皮基本的结构和功能单位,恒定地暴露于血流中,其表面受体易受到多种因素的影响,可使VEC合成、表达炎症和凝血相关的因子。补体活化产物C5a在脓毒症及其并发的MODS发病过程中,既可通过趋化白细胞和激活炎症效应细胞表达促炎分子参与全身炎症反应;也可直接激活炎症靶细胞VEC、中性粒细胞(PMN),促使其表达多种粘附分子,诱导PMN与VEC黏附和跨膜。同时,C5a也可刺激VEC产生组织因子而参与血管内凝血的启动,在局部C5a还能调节炎症介质的表达、促进趋化PMN聚集、诱导PMN脱颗粒,造成VEC的炎症损伤,这些均成为C5a在脓毒症中引起MODS的病理基础。而C5a参与以上组织炎症反应、损伤及微循环障碍主要通过炎症细胞的C5a受体(C5aR)介导;阻断C5a与C5aR的相互作用有望抑制其产生的炎症反应、损伤及微循环障碍的病理过程。血栓调节素(TM,CD141)是血管VEC合成的一种膜表面糖蛋白,是VEC表面的凝血酶受体之一,在抗凝血与抗炎方面都有着重要作用,而凝血、炎症又是脓毒症脏器损伤的病理基础,同时C5a也是诱发脓毒症的原因之一。因此,探讨C5a对TM在VEC上的表达的影响及其机制可以为指导脓毒症的治疗提供一个新的方向。目的:探讨补体C5a对人脐静脉VEC(HUVECs)血栓调节蛋白(TM)表达的影响。方法:通过建立HUVEC体外培养,观察1、以终浓度200 ug/L重组人C5a(rh-C5a)刺激HUVECs 8、12、16、20 h以及以终浓度100、200、300ug/L的C5a刺激HUVECs 12 h;2、以终浓度为200ug/L的C5a分别刺激HUVECs 4、8、12h时即用自主合成的C5aR反义肽(R4)以终浓度为2mg/L进行干预,以16小时作为刺激终点;3、以终浓度为2ug/ml的BAY 11-7082[核因子-κB(NF-κB)的抑制剂]在37℃孵育1小时后,再以终浓度为200ng/ml的C5a作用于HUVECs 12小时。采用实时荧光定量PCR、蛋白免疫印迹法(Western blot)分别检测TM的mRNA及蛋白表达变化;观察C5a对其表达TM的量-效和时-效关系、R4的拮抗效能及NF-κB信号通路的作用。结果:1、C5a抑制了HUVECs在TM的mRNA和细胞膜表面蛋白水平表达。同时,C5a对TM表达的抑制作用存在量-效关系(蛋白比(106):1.1325±0.0397,0.8018±0.0256,0.7322±0.0436;mRNA比(10-4):4.0177±0.2046,0.3611±0.0351,0.1819±0.0146,P均<0.05)和时-效关系(蛋白比:0.9311±0.01567,0.7105±0.03391,0.6548±0.04285,0.6269±0.04031;mRNA(10-4):3.0171±0.8040,0.3829±0.2024,0.0882±0.0027,0.0705±0.0080,P均<0.05);TM的mRNA水平于200 ug/L C5a刺激12 h后降低程度明显减缓,蛋白水平于300 ug/L C5a刺激12 h后降低程度明显减缓。2、在mRNA和细胞膜表面蛋白水平, R4均能减少C5a降低HUVECs表达TM的程度,且越早进行干预其效果越明显。3、NF-κB信号通路被阻断后,减弱了C5a对HUVECs在TM的mRNA和细胞膜表面蛋白水平表达的抑制作用。结论:1、C5a刺激可抑制HUVECs的TM结构基因表达,进而减弱TM的蛋白翻译,从而参与了脓毒症时凝血亢进、炎症损害的病理生理过程。2、应用C5a拮抗多肽能减轻C5a对HUVECs表达TM的抑制程度,提示阻断C5a与C5aR的结合可改善C5a参与了脓毒症时凝血亢进、炎症损害的病理生理过程。3、应用NF-κB抑制剂能减少C5a对HUVECs表达TM的抑制程度,提示TM表达受NF-κB信号通路调控。
【Abstract】 The mechanism of Multiple organ dysfunction syndrome(MODS) lesded by systemic inflammatory response syndrome(SIRS) is associated with uncontrolled host defense responses that lead to inflammation, endothelial damage, enhanced coagulation and diminished fibrinolysis. Vein endothelial cells (VEC) is the base of blood vessel endothelium, they always exposed in blood, and their various kinds of receptors on the surface were influenced by different factors, and lead VECs to produce and express various kinds of inflammation mediator which are associated with inflammation and coagulation.The C5a do its job both at SIRS and MODS,it not only can reglaution polymorphonuclear neutrophil (PMN) motion and activate inflammation cells to express promoptor factors to join systemic inflammatory response, but also can activate target cell, such as VECs and PMN, lead them to express lots of adhesion molecule, and then leads them activate,adhesion,traval to outside of blood vessel. At another side , C5a can activate VECs to express tissues factor and leads to coagulation in vessels. At partly of organ, C5a can regulate the expression of inflammation mediator, promopt PMN motion and assemble and injury VECs. All of this are pathological base of C5a works in MODS. C5a induce all of injury is based on combining with its receptor C5aR, blocking the combining with C5a and C5aR, perhaps, can restrain the pathological process which can be leaded by C5a.Thrombomodulin (TM,CD141) is a kind of glycoprotein producted by VECs on its surface, it is one of the receptors of thrombin, too.TM is a important role both at anti-coagulation and at anti- inflammation,and coagulation and inflammation are pathological base of MODS in SIRS. Another side, C5a is one of the reasons inducing SIRS. So by this study, we can know the regulation of VECs express TM by C5a and its mechanism,by this means, we can provide a new method for treat with SIRS.Objective:To investigate the effect of C5a on the express of thrombomodulin of Human umbilical vein endothelial cells (HUVECs).Methods: 1,HUVECs cultured in vitro were stimulated with C5a in 8,12,16,20 hours by concentration of 200 ng/ml, and by different concentrations of 100、200、300ng/ml in 12 hours respectively. 2,HUVECs cultured in vitro were stimulated by concentration of C5a with 200 ng/ml at 4h,8h,12h, and then interfered by antagonism polypeptide of C5a receptor(R4) with 2mg/L,at the end by 16h. 3,Use inhibitor of NF-κB (BAY 11-7082)by concentration of 2ug/ml culture HUVECs for 1h, and then stimulated by concentration of C5a with 200 ng/ml for 12h.TM expressions at mRNA levels and protein levels was detected by Real-time PCR and Western-blot respectively, and the dose and time depended effect of C5a on the expression of TM is evaluated.Results: 1,C5a down-regulates the TM expression at both protein and mRNA level. The down-regulation is time-dependence(1.1325±0.0397,0.8018±0.0256,0.7322±0.0436 at protein level and 4.0177±0.2046,0.3611±0.0351,0.1819±0.0146 (10-4)at mRNA level,both of them p<0.05) and dose-dependence : (0.9311±0.0157,0.7105±0.0339,0.6548±0.0429,0.6269±0.0403 at protein level and 3.0171±0.8040,0.3829±0.2024,0.0882±0.0027,0.0705±0.0080 (10-4) at mRNA level,both of them p<0.05).When concentration of C5a at 200 ng/ml is used to stimulate HUVECs for 12 hours, the underspeed of TM at mRNA level will slow down obviously;concentration of C5a at 300 ng/ml is used to stimulate HUVECs for 12 hours, the underspeed of TM at protein level will slow down obviously. 2,R4 can decrease the level of regulation of HUVEs express TM by C5a at protein and mRNA level, and the earlier interfered by R4 the decreasing of the down-regulation is more obviously. 3, NF-κB pathway is blocked, both at mRNA and protein level, the down-regulation of TM by C5a is decrease.Conclusions:1,C5a can depress the gene expression of TM, and then effect the protein’s translation. By this means , C5a can lead to coagulation-accentuation and inflammation-injuries in sepsis.2, Block the combination between C5a and C5aR, the down-regulation of TM by C5a is decrease. This reveal that blocking the combining of C5a and C5aR can decrease the injury of coagulation-accentuation and inflammation-injuries in sepsis.3, Inhibit the of NF-κB can decrease the down-regulation of TM by C5a, This reveal that NF-κB pathway can regulate the express of TM.