本文選題：C5a + 臍靜脈VEC��； 參考：《廣州醫(yī)學(xué)院》2009年碩士論文

【摘要】： 膿毒癥導(dǎo)致多臟器功能障礙綜合征(MODS)的發(fā)病機(jī)理主要與失控的宿主防御反應(yīng)導(dǎo)致炎癥、內(nèi)皮損傷、凝血亢進(jìn)、纖溶減弱等有關(guān)。血管內(nèi)皮細(xì)胞(VEC)是血管內(nèi)皮基本的結(jié)構(gòu)和功能單位,恒定地暴露于血流中,其表面受體易受到多種因素的影響,可使VEC合成、表達(dá)炎癥和凝血相關(guān)的因子。 補(bǔ)體活化產(chǎn)物C5a在膿毒癥及其并發(fā)的MODS發(fā)病過程中,既可通過趨化白細(xì)胞和激活炎癥效應(yīng)細(xì)胞表達(dá)促炎分子參與全身炎癥反應(yīng);也可直接激活炎癥靶細(xì)胞VEC、中性粒細(xì)胞(PMN),促使其表達(dá)多種粘附分子,誘導(dǎo)PMN與VEC黏附和跨膜。同時(shí),C5a也可刺激VEC產(chǎn)生組織因子而參與血管內(nèi)凝血的啟動(dòng),在局部C5a還能調(diào)節(jié)炎癥介質(zhì)的表達(dá)、促進(jìn)趨化PMN聚集、誘導(dǎo)PMN脫顆粒,造成VEC的炎癥損傷,這些均成為C5a在膿毒癥中引起MODS的病理基礎(chǔ)。而C5a參與以上組織炎癥反應(yīng)、損傷及微循環(huán)障礙主要通過炎癥細(xì)胞的C5a受體(C5aR)介導(dǎo);阻斷C5a與C5aR的相互作用有望抑制其產(chǎn)生的炎癥反應(yīng)、損傷及微循環(huán)障礙的病理過程。 血栓調(diào)節(jié)素(TM,CD141)是血管VEC合成的一種膜表面糖蛋白,是VEC表面的凝血酶受體之一,在抗凝血與抗炎方面都有著重要作用,而凝血、炎癥又是膿毒癥臟器損傷的病理基礎(chǔ),同時(shí)C5a也是誘發(fā)膿毒癥的原因之一。因此,探討C5a對(duì)TM在VEC上的表達(dá)的影響及其機(jī)制可以為指導(dǎo)膿毒癥的治療提供一個(gè)新的方向。 目的:探討補(bǔ)體C5a對(duì)人臍靜脈VEC(HUVECs)血栓調(diào)節(jié)蛋白(TM)表達(dá)的影響。 方法:通過建立HUVEC體外培養(yǎng),觀察1、以終濃度200 ug/L重組人C5a(rh-C5a)刺激HUVECs 8、12、16、20 h以及以終濃度100、200、300ug/L的C5a刺激HUVECs 12 h;2、以終濃度為200ug/L的C5a分別刺激HUVECs 4、8、12h時(shí)即用自主合成的C5aR反義肽(R4)以終濃度為2mg/L進(jìn)行干預(yù),以16小時(shí)作為刺激終點(diǎn);3、以終濃度為2ug/ml的BAY 11-7082[核因子-κB(NF-κB)的抑制劑]在37℃孵育1小時(shí)后,再以終濃度為200ng/ml的C5a作用于HUVECs 12小時(shí)。采用實(shí)時(shí)熒光定量PCR、蛋白免疫印跡法(Western blot)分別檢測TM的mRNA及蛋白表達(dá)變化;觀察C5a對(duì)其表達(dá)TM的量-效和時(shí)-效關(guān)系、R4的拮抗效能及NF-κB信號(hào)通路的作用。 結(jié)果:1、C5a抑制了HUVECs在TM的mRNA和細(xì)胞膜表面蛋白水平表達(dá)。同時(shí),C5a對(duì)TM表達(dá)的抑制作用存在量-效關(guān)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)和時(shí)-效關(guān)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后降低程度明顯減緩,蛋白水平于300 ug/L C5a刺激12 h后降低程度明顯減緩。2、在mRNA和細(xì)胞膜表面蛋白水平, R4均能減少C5a降低HUVECs表達(dá)TM的程度,且越早進(jìn)行干預(yù)其效果越明顯。3、NF-κB信號(hào)通路被阻斷后,減弱了C5a對(duì)HUVECs在TM的mRNA和細(xì)胞膜表面蛋白水平表達(dá)的抑制作用。 結(jié)論: 1、C5a刺激可抑制HUVECs的TM結(jié)構(gòu)基因表達(dá),進(jìn)而減弱TM的蛋白翻譯,從而參與了膿毒癥時(shí)凝血亢進(jìn)、炎癥損害的病理生理過程。 2、應(yīng)用C5a拮抗多肽能減輕C5a對(duì)HUVECs表達(dá)TM的抑制程度,提示阻斷C5a與C5aR的結(jié)合可改善C5a參與了膿毒癥時(shí)凝血亢進(jìn)、炎癥損害的病理生理過程。 3、應(yīng)用NF-κB抑制劑能減少C5a對(duì)HUVECs表達(dá)TM的抑制程度,提示TM表達(dá)受NF-κB信號(hào)通路調(diào)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.

【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392.12

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 陳月;李保勝;李元朝;呂鳳林;胡承香;;C5a反義肽對(duì)膿毒癥小鼠的保護(hù)作用及其機(jī)制[J];中華實(shí)驗(yàn)外科雜志;2005年12期

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本文編號(hào)：1827966