C5a对血管内皮细胞表达血栓调节蛋白的影响

发布时间：2018-05-01 05:32

  本文选题：C5a + 脐静脉VEC　； 参考：《广州医学院》2009年硕士论文

【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]:Sepsis leads to the pathogenesis of multiple organ dysfunction syndrome (MODS), which is mainly associated with an uncontrolled host defense response to inflammation, endothelial damage, hyperactivity of coagulation, and fibrinolysis. Vascular endothelial cells (VEC) are the basic structural and functional units of vascular endothelium, which are invariably exposed to the blood flow, and their surface receptors are susceptible to a variety of factors. It can make VEC synthesis and express inflammatory and coagulation related factors.
In the pathogenesis of sepsis and its concurrent MODS, the complement activation product C5a can not only express the inflammatory response through the chemotaxis of leukocytes and activate inflammatory cells, but also directly activate the inflammatory target cells VEC, neutrophils (PMN), promote the expression of multiple adhesion molecules, induce the adhesion and transmembrane of PMN and VEC, and C5a It also stimulates VEC to produce tissue factors and participate in the initiation of intravascular coagulation. Local C5a can also regulate the expression of inflammatory mediators, promote chemotactic PMN aggregation, induce PMN degranulation and cause VEC inflammatory damage. These all become the pathological basis of C5a in sepsis caused by MODS, and C5a participates in the inflammatory response, injury and microcirculation disorders in the above tissues. It is mainly mediated by the C5a receptor (C5aR) of inflammatory cells, and blocking the interaction between C5a and C5aR may inhibit the inflammatory reaction, injury and the pathological process of microcirculation disorder.
TM (CD141) is a membrane surface glycoprotein synthesized by VEC and is one of the thrombin receptors on the surface of VEC. It plays an important role in anticoagulant and anti-inflammatory. And coagulation, inflammation is the pathological basis of sepsis, and C5a is also one of the causes of sepsis. Therefore, the C5a table on TM in VEC is discussed. The effect and mechanism of DA can provide a new direction for the treatment of sepsis.
Objective: To investigate the effect of complement C5a on the expression of thrombomodulin (TM) in human umbilical vein VEC (HUVECs).
Methods: HUVEC was cultured in vitro, and 1 was observed. HUVECs 8,12,16,20 h was stimulated with a final concentration of 200 ug/L recombinant human C5a (rh-C5a), and HUVECs 12 h was stimulated with the final concentration of 100200300ug/L, and 2. At 16 hours, 3, after incubating the final concentration of BAY 11-7082[nuclear factor kappa B (NF- kappa B) for 1 hours at 37 degrees, the final concentration of 200ng/ml C5a acted on HUVECs 12 hours. Real time fluorescent quantitative PCR and protein immunoblotting (Western blot) were used to detect the changes of protein expression and protein expression, respectively. The expression of TM was related to the dose effect and time effect relationship, the antagonistic efficacy of R4 and the role of NF- kappa B signaling pathway.
Results: 1, C5a inhibited the expression of HUVECs in TM mRNA and cell membrane surface protein level. At the same time, the inhibitory effect of C5a on the expression of TM has a quantitative effect relationship (protein ratio (106): 1.1325 + 0.0397,0.8018 + 0.0256,0.7322 + 0.0436; mRNA ratio (10-4): 4.0177 + 0.2046,0.3611 + 0.0351,0.1819 + 0.0146, P are 0.05) and time effect relationship (protein ratio: 0.9311 + 0.01) 567,0.7105 + 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 0.05); TM mRNA level was significantly slowed down after 200 ug/L C5a stimulated 12, protein level was slowed down after 12 stimulating 12. Level, R4 can reduce the degree of C5a to reduce HUVECs expression of TM, and the earlier the intervention its effect is more obvious.3, after the NF- kappa B signal pathway is blocked, the inhibitory effect of C5a on HUVECs in TM mRNA and the expression of protein level on the cell membrane surface is weakened.
Conclusion:
1, C5a stimulation inhibits the TM structural gene expression of HUVECs and then weakens the protein translation of TM, thus participating in the pathophysiological process of hypercoagulability and inflammatory damage in sepsis.
2, the application of C5a antagonist peptide can reduce the inhibition of C5a on HUVECs expression of TM, suggesting that blocking the combination of C5a and C5aR can improve the pathophysiological process of C5a involved in hypercoagulability and inflammatory damage in sepsis.
3, the application of NF- kappa B inhibitor can reduce the inhibition of C5a on HUVECs expression of TM, suggesting that TM expression is regulated by NF- NF- B signal pathway.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.12

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