TLR4在抗β2GPI抗体诱导小鼠血管黏附分子及组织因子表达中的作用探讨

发布时间：2018-05-17 23:44

本文选题：抗磷脂综合征 + TLR4　；参考：《江苏大学》2015年硕士论文

【摘要】：研究目的：抗磷脂综合征(antiphospholipid syndrome, APS)主要临床症状包括血清中存在的高滴度抗磷脂抗体(antiphospholipid antibodies, aPLs)、反复发作的动静脉血栓形成、血小板减少、习惯性流产等。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被认为在血栓形成中起到重要作用。抗β2GPI抗体(anti-β2GPI Ab)是与APS患者血栓形成关系最为密切的aPL,阴性磷脂结合蛋白β2糖蛋白I (β2-glycoprotein I, p2GPI)是抗β2GPI抗体的关键靶抗原。有大量文献表明,抗β2GPI抗体(anti-β2GPI Abs)可以通过TLR4/NF-KB通路引起内皮细胞黏附分子(ICAM-1、ICAM-1、E-selectin)及组织因子(tissue factor, TF)的高表达。但是,对抗β2GPI抗体在体内引起内皮细胞功能异常的机制仍旧缺乏足够的研究。基于这一背景,我们利用抗β2GPI抗体刺激C3H/HeN小鼠(TLR4表达正常)以及C3H/HeJ小鼠(TLR4表达缺陷),探讨TLR4及其下游信号分子p38MAPK和NF-κB p65是否参与抗β2GPI抗体介导的小鼠血管内皮细胞活化和黏附分子与组织因子的表达。研究方法：(1)分别用抗β2GPI抗体、LPS和生理盐水通过腹腔注射刺激C3H/HeN及C3H/HeJ两种小鼠。0 h、48 h各注射一次,72 h后取小鼠动脉进行免疫组织化学染色,检测小鼠动脉内皮细胞层中VCAM-1、ICAM-1、E-selectin蛋白表达情况,观察抗β2GPI抗体对小鼠内皮细胞的活化作用及TLR4在其中的作用。(2)利用Western blot及实时荧光定量PCR (Real-time quantitative PCR, RT-qPCR)检测抗β2GPI抗体、LPS及生理盐水组刺激后C3H/HeN和C3H/HeJ小鼠动脉血管匀浆中的ICAM-1、VCAM-1、E-selectin蛋白及mRNA的表达水平。(3)抗p2GPI抗体刺激C3H/HeN及C3H/HeJ两种小鼠后,利用RT-qPCR和TF活性试剂盒检测主动脉血管匀浆中TF的mRNA表达和蛋白活性,以观察TLR4对动脉血管的TF表达的影响。(4)利用Western blot检测C3H/HeN和C3H/HeJ小鼠分别经anti-β2GPI、LPS、生理盐水刺激后,动脉血管匀浆中p38MAPK以及NF-κB p65的总蛋白及磷酸化水平,以观察anti-β2GPI对小鼠动脉TLR4/NF-KB通路的影响。研究结果(1)经抗β2GPI抗体刺激后,C3H/HeN小鼠的动脉内皮细胞层ICAM-1、 VCAM-1、E-selectin分子的阳性染色率较生理盐水组明显增高,与LPS组具有相同的刺激效应。而C3H/HeJ小鼠经anti-β2GPI刺激后,动脉内皮未见明显阳性表达,与相同刺激的C3H/HeN小鼠相较有显著差异。(2)经抗β2GPI抗体刺激后,C3H/HeN小鼠动脉血管匀浆中VCAM-1、 ICAM-1和E-selectin mRNA和蛋白表达升高,与生理盐水组或C3H/HeJ小鼠刺激组相比差异显著,且具有统计学意义(p0.05)(3)抗β2GPI抗体刺激后,C3H/HeN小鼠动脉匀浆中TF的mRNA水平和蛋白活性高于C3H/HeJ小鼠,该差异具有统计学意义(p0.05)(4)抗β2GPI抗体刺激后,C3H/HeN小鼠动脉血管匀浆中p38 MAPK和NF-κB p65的磷酸化水平升高,并且与生理盐水组或C3H/HeJ小鼠刺激组差异显著(p0.05)。结论：(1)抗β2GPI抗体能够在体内引起小鼠血管内皮细胞的活化,从而高表达粘附分子与组织因子,这与之前的体外实验的报道是一致的。(2)TLR4基因的缺陷可显著但不完全地抑制抗β2GPI抗体对内皮细胞的活化,这一结果提示抗β2GPI抗体诱导的内皮细胞活化可能部分依赖于TLR4的作用：(3)抗β2GPI抗体刺激后引起的TLR4下游信号分子p38MAPK、 NF-κB p65高表达和磷酸化提示TLR4/NF-κB信号通路可能参与到抗β2GPI抗体诱导的小鼠血管内皮活化。
[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.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.2

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