本文選題：抗磷脂綜合征 + TLR4��； 參考：《江蘇大學》2015年碩士論文

【摘要】：研究目的：抗磷脂綜合征(antiphospholipid syndrome, APS)主要臨床癥狀包括血清中存在的高滴度抗磷脂抗體(antiphospholipid antibodies, aPLs)、反復發(fā)作的動靜脈血栓形成、血小板減少、習慣性流產(chǎn)等。其中,血栓是APS病人致死的主要原因之一,而血清中高滴度的aPLs被認為在血栓形成中起到重要作用�？功�2GPI抗體(anti-β2GPI Ab)是與APS患者血栓形成關系最為密切的aPL,陰性磷脂結(jié)合蛋白β2糖蛋白I (β2-glycoprotein I, p2GPI)是抗β2GPI抗體的關鍵靶抗原。有大量文獻表明,抗β2GPI抗體(anti-β2GPI Abs)可以通過TLR4/NF-KB通路引起內(nèi)皮細胞黏附分子(ICAM-1、ICAM-1、E-selectin)及組織因子(tissue factor, TF)的高表達。但是,對抗β2GPI抗體在體內(nèi)引起內(nèi)皮細胞功能異常的機制仍舊缺乏足夠的研究�；谶@一背景,我們利用抗β2GPI抗體刺激C3H/HeN小鼠(TLR4表達正常)以及C3H/HeJ小鼠(TLR4表達缺陷),探討TLR4及其下游信號分子p38MAPK和NF-κB p65是否參與抗β2GPI抗體介導的小鼠血管內(nèi)皮細胞活化和黏附分子與組織因子的表達。研究方法：(1)分別用抗β2GPI抗體、LPS和生理鹽水通過腹腔注射刺激C3H/HeN及C3H/HeJ兩種小鼠。0 h、48 h各注射一次,72 h后取小鼠動脈進行免疫組織化學染色,檢測小鼠動脈內(nèi)皮細胞層中VCAM-1、ICAM-1、E-selectin蛋白表達情況,觀察抗β2GPI抗體對小鼠內(nèi)皮細胞的活化作用及TLR4在其中的作用。(2)利用Western blot及實時熒光定量PCR (Real-time quantitative PCR, RT-qPCR)檢測抗β2GPI抗體、LPS及生理鹽水組刺激后C3H/HeN和C3H/HeJ小鼠動脈血管勻漿中的ICAM-1、VCAM-1、E-selectin蛋白及mRNA的表達水平。(3)抗p2GPI抗體刺激C3H/HeN及C3H/HeJ兩種小鼠后,利用RT-qPCR和TF活性試劑盒檢測主動脈血管勻漿中TF的mRNA表達和蛋白活性,以觀察TLR4對動脈血管的TF表達的影響。(4)利用Western blot檢測C3H/HeN和C3H/HeJ小鼠分別經(jīng)anti-β2GPI、LPS、生理鹽水刺激后,動脈血管勻漿中p38MAPK以及NF-κB p65的總蛋白及磷酸化水平,以觀察anti-β2GPI對小鼠動脈TLR4/NF-KB通路的影響。研究結(jié)果(1)經(jīng)抗β2GPI抗體刺激后,C3H/HeN小鼠的動脈內(nèi)皮細胞層ICAM-1、 VCAM-1、E-selectin分子的陽性染色率較生理鹽水組明顯增高,與LPS組具有相同的刺激效應。而C3H/HeJ小鼠經(jīng)anti-β2GPI刺激后,動脈內(nèi)皮未見明顯陽性表達,與相同刺激的C3H/HeN小鼠相較有顯著差異。(2)經(jīng)抗β2GPI抗體刺激后,C3H/HeN小鼠動脈血管勻漿中VCAM-1、 ICAM-1和E-selectin mRNA和蛋白表達升高,與生理鹽水組或C3H/HeJ小鼠刺激組相比差異顯著,且具有統(tǒng)計學意義(p0.05)(3)抗β2GPI抗體刺激后,C3H/HeN小鼠動脈勻漿中TF的mRNA水平和蛋白活性高于C3H/HeJ小鼠,該差異具有統(tǒng)計學意義(p0.05)(4)抗β2GPI抗體刺激后,C3H/HeN小鼠動脈血管勻漿中p38 MAPK和NF-κB p65的磷酸化水平升高,并且與生理鹽水組或C3H/HeJ小鼠刺激組差異顯著(p0.05)。結(jié)論：(1)抗β2GPI抗體能夠在體內(nèi)引起小鼠血管內(nèi)皮細胞的活化,從而高表達粘附分子與組織因子,這與之前的體外實驗的報道是一致的。(2)TLR4基因的缺陷可顯著但不完全地抑制抗β2GPI抗體對內(nèi)皮細胞的活化,這一結(jié)果提示抗β2GPI抗體誘導的內(nèi)皮細胞活化可能部分依賴于TLR4的作用：(3)抗β2GPI抗體刺激后引起的TLR4下游信號分子p38MAPK、 NF-κB p65高表達和磷酸化提示TLR4/NF-κB信號通路可能參與到抗β2GPI抗體誘導的小鼠血管內(nèi)皮活化。
[Abstract]:Research objectives: the main clinical symptoms of antiphospholipid syndrome (APS) include the high titer anti phospholipid antibody (antiphospholipid antibodies, aPLs) in the serum, the recurrent arteriovenous thrombosis, thrombocytopenia, and habitual abortion. Among them, thrombosis is one of the main causes of death in APS patients, and blood is one of the main causes of death. The high titer of aPLs is considered to play an important role in the formation of thrombus. Anti beta 2GPI antibody (anti- beta 2GPI Ab) is the most closely related aPL in the thrombosis of APS patients, and the negative phospholipid binding protein beta 2 glycoprotein I (beta 2-glycoprotein I, p2GPI) is the key target antigen for the anti beta 2GPI antibody. GPI Abs) can cause high expression of endothelial cell adhesion molecules (ICAM-1, ICAM-1, E-selectin) and tissue factors (tissue factor, TF) through the TLR4/NF-KB pathway. However, there is still a lack of research on the mechanism of the abnormal function of endothelial cells caused by beta 2GPI antibodies in the body. Based on this background, we use anti beta 2GPI antibodies to stimulate C3H/HeN. Mice (TLR4 expression) and C3H/HeJ mice (TLR4 expression deficiency) were used to investigate whether TLR4 and its downstream signal molecules p38MAPK and NF- kappa B p65 were involved in the expression of adhesion molecules and tissue factors in mice vascular endothelial cells mediated by anti beta 2GPI antibody. Methods: (1) the anti beta 2GPI antibody, LPS and physiological saline were injected into the abdominal cavity, respectively. Two mice of C3H/HeN and C3H/HeJ were injected into.0 h, 48 h were injected once, and the mice artery was stained by immunohistochemistry after 72 h. The expression of VCAM-1, ICAM-1 and E-selectin protein in the endothelial cell layer of the mice was detected. The effect of anti beta 2GPI antibody on the survival of mice endothelial cells was observed and the role of TLR4 in the mice was observed. (2) utilize Western. And real-time fluorescent quantitative PCR (Real-time quantitative PCR, RT-qPCR) for the detection of anti beta 2GPI antibody, ICAM-1, VCAM-1, E-selectin protein and expression level in the arterial homogenate of C3H/HeN and C3H/HeJ mice after LPS and normal saline group. (3) after the anti antibody stimulation and two kinds of mice, the activity test was used. The expression of mRNA and protein activity of TF in aortic vascular homogenate were detected by the agent box to observe the effect of TLR4 on the expression of TF in arterial blood vessels. (4) Western blot was used to detect the total protein and phosphorylation level in the arterial homogenate of C3H/HeN and C3H/HeJ mice after the anti- beta 2GPI, LPS, and physiological saline stimulation. The effect of ti- beta 2GPI on TLR4/NF-KB pathway in mouse artery. Results (1) after the stimulation of anti beta 2GPI antibody, the positive staining rate of ICAM-1, VCAM-1, E-selectin molecules in the arterial endothelial layer of C3H/HeN mice was significantly higher than that in the saline group, and the same stimulation effect was found in the LPS group. And the C3H/HeJ mice were stimulated by anti- beta 2GPI. No significant positive expression was found in the C3H/HeN mice with the same stimulation. (2) the expression of VCAM-1, ICAM-1 and E-selectin mRNA and protein in the arterial homogenate of C3H/HeN mice increased after the stimulation of anti beta 2GPI antibody, which was significantly different from that of the saline group or the C3H/HeJ mice stimulation group, and was statistically significant (P0.05) (3) anti beta 2. After the stimulation of GPI antibody, the mRNA level and protein activity of TF in the arterial homogenate of C3H/HeN mice were higher than that of C3H/HeJ mice. The difference was statistically significant (P0.05) (4) the phosphorylation level of p38 MAPK and NF- kappa B in the arterial homogenate of C3H/HeN mice increased, and was different from the saline group or the mice stimulation group. Significant (P0.05). Conclusion: (1) anti beta 2GPI antibody can induce the activation of vascular endothelial cells in mice in vivo, thus high expression of adhesion molecules and tissue factors, which is consistent with the previous reports in vitro. (2) the defect of TLR4 gene can significantly but not completely inhibit the activation of anti beta 2GPI antibody to endothelial cells. This result is a result suggested. The activation of endothelial cells induced by anti beta 2GPI antibody may be partly dependent on the effect of TLR4: (3) the downstream signal molecule p38MAPK of TLR4 induced by anti beta 2GPI antibody, NF- kappa B p65 high expression and phosphorylation suggest that TLR4/NF- kappa B signaling pathway may be involved in the activation of vascular endothelial cells induced by anti beta 2GPI antibody.
【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R593.2

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