【作者】 王婷；

【导师】 周红；

【作者基本信息】 江苏大学 ， 临床检验诊断学， 2013， 博士

【摘要】 研究目的：β2糖蛋白I (beta-2-glycoproteinI, β2GPI)与其相应抗体(抗β2GPI, anti-β2GPI)形成复合物,能够刺激血液单核细胞、血管内皮细胞活化,表达相关细胞因子,参与抗磷脂综合征(antiphospholipid syndrome, APS)的高凝状态和炎性反应,是其重要的病理机制之一。课题组前期研究揭示,在anti-β2GPI/β2GPI复合物刺激人单核细胞株THP-1表达组织因子(tissue factor, TF)的过程中,跨膜蛋白Toll样受体4(Toll-like receptor4, TLR4)及其下游信号转导分子能够被诱导表达并参与细胞的激活作用,并且部分依赖膜联蛋白Ⅱ (Annexin A2, ANX2),成为APS血栓形成的机制之一。本论文旨在前期研究基础上,以TF和肿瘤坏死因子-α (tumor necrosis factor, TNF-a)为指标,探讨绿茶中重要的活性成分表没食子儿茶素-3-没食子酸酯(epigallocatechin-3-gallate, EGCG)以及他汀类之一的氟伐他汀能否抑制mti-β2GPI/β2GPI复合物介导的单核细胞株THP-1活化,其具体分子机制如何,为APS预防和治疗提供新的思路。研究方法：(1)利用实时定量PCR (Real-time quantitative PCR, RT-qPCR)检测anti-β2GPI/β2GPI复合物诱导THP-1细胞TF和TNF-α mRNA的表达,采用试剂盒检测TF活性,ELISA试剂盒检测TNF-a蛋白含量。(2)采用不同浓度的EGCG和氟伐他汀预处理THP-1细胞,MTT法观察对细胞增殖活力的影响。(3)观察不同浓度的EGCG和氟伐他汀对anti-β2GPI/β2GPI复合物诱导THP-1细胞表达TF和TNF-a的抑制作用,并检测其量效性和特异性。(4)运用RT-qPCR和Western blotting方法检测EGCG和氟伐他汀对anti-β2GPI/β2GPI复合物诱导的THP-1细胞TLR4mRNA和蛋白表达的抑制作用。(5) Western blotting检测anti-β2GPI/β2GPI复合物刺激THP-1细胞有丝分裂原蛋白激酶(mitogen-activated protein kinases, MAPKs)与核因子κB (nuclear factor kappa B, NF-κB)的活化情况,并检测刺激的时效性。(6)采用不同浓度的EGCG和氟伐他汀预处理THP-1细胞,观察其对anti-β/β2GPI复合物诱导THP-1细胞MAPKs和NF-κB p65表达和磷酸化的干预作用,并检测其量效性。研究结果：(1) Anti-β2GPI/β2GPI复合物能够刺激THP-1细胞表达TF (mRNA及活性)和TNF-α (mRNA及蛋白),与对照组相比,差异显著(p<0.05)。(2)较低浓度的EGCG和氟伐他汀不影响THP-1细胞增殖活力。当EGCG浓度为100μg/ml,氟伐他汀浓度为50μg/ml时,可以显著抑制THP-1细胞增殖(p<0.05)。(3) EGCG和氟伐他汀抑制anti-β2GPI/β2GPI复合物对THP-1细胞TF和TNF-α表达的刺激效应,并呈现浓度依赖性。(4) Anti-β2GPI/β2GPI复合物刺激THP-1细胞后,TLR4表达增高；EGCG和氟伐他汀可以抑制anti-β2GPI/β2GPI复合物诱导的THP-1细胞TLR4mRNA和蛋白表达。(5) Anti-β2GPI/β2GPI复合物刺激THP-1细胞表达TF和TNF-α,同时增加THP-1细胞MAPKs和NF-κB的磷酸化水平,并显示时间效应。EGCG和氟伐他汀显著抑制anti-β2GPI/β2GPI复合物诱导的THP-1细胞MAPKs (ERK1/2、JNK、p38)和NF-κB的磷酸化(p<0.05),并呈现一定的浓度依赖性。结论：(1) Anti-β2GPI/β2GPI复合物能够刺激单核细胞THP-1活化,使细胞表达TF及TNF-α显著增加。(2) EGCG和氟伐他汀能抑制anti-β2GPI/β2GPI复合物对THP-1细胞TF和TNF-α表达的刺激效应。(3) EGCG和氟伐他汀干预anti-β2GPI/β2GPI复合物刺激的TLR4表达增强,以及MAPKs和NF-κB的磷酸化。(4) EGCG和氟伐他汀通过抑制TLR4-MAPKs-NF-κB信号通路,干预anti-β2GPI/β2GPI复合物对THP-1细胞的活化,提示可用于APS血栓形成的预防及治疗。更多还原

【Abstract】 Objective:Anti-β2-glycoproteinI/β2-glycoproteinI (anti-β2GPI/β2GPI) complex could activate endothelial cells and monocytes upon binding to the surface membrane of the cells, enhancing the expression and secretion of many cytokines, thereby increasing the risk of thrombosis, which are beneficial to the thrombus formation of antiphospholipid syndrome (APS). Our previous study reveals that the Toll-like receptor4(TLR4) and its signal transduction pathway can be induced to express and participate in cellular activation, and the effects of TLR4with annexinA2(ANX2) are tightly cooperative in process of anti-β2GPI/β2GPI-stimulated tissue factor (TF) and tumor necrosis factor-a (TNF-a) expression in the human monocytic cell line THP-1. These may be responsible for the thrombotic mechanisms in APS. In the present study, we investigated the effects of EGCG (epigallocatechin-3-gallate) and fluvastatin on anti-β2GPI/β2GPI complex-induced THP-1cells activation, targeting to the expression of TF and TNF-a, as well as the possible mechanisms involved in this process. The study may provide a new way for prevention and treatment of thrombotic issues in APS.Methods:(1) The THP-1cells were treated with anti-β2GPI/β2GPI complex. The TF and TNF-a mRNA expression on cells were detected by Real-time quantitative PCR (RT-qPCR), the TF activity and TNF-α protein level were estimated by commercial kits respectively.(2) Pretreated with different concentration of EGCG or fluvastatin, the cell proliferation ability was detected by MTT assay. (3) Pretreated with different concentration of EGCG or fluvastatin, the levels of TF and TNF-a induced by anti-β2GPI/β2GPI complex were examined by RT-qPCR and commercial kits respectively. The dose course and specific effects of EGCG or fluvastatin on THP-1cells were also investigated.(4) EGCG and fluvastatin were used to investigate whether the TLR4expression (mRNA and protein) in cells induced by anti-β2GPI/β2GPI could be intervened.(5) The phosphorylation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) in THP-1cells induced by anti-β2GPI/β2GPI complex were investigated by western blotting. The time course of anti-β2GPI/β2GPI complex in cells was also investigated.(6) EGCG and fluvastatin were used to investigate whether the phosphorylation of NF-κB and MAPKs in cells induced by anti-β2GPI/β2GPI could be decreased by western blot assays, the dose course in THP-1cells was also investigated.Results:(1) The TF and TNF-α expression in THP-1cells were significantly up-regulated with treatment of anti-β2GPI/β2GPI complex, compared with untreated cells (p<0.05).(2) High concentration of EGCG (100μg/ml) or fluvastatin (50μg/ml) could inhibit cell proliferation in THP-1cells.(3) EGCG and fluvastatin could dose-dependently decrease TF and TNF-a level in THP-1cells stimulated with anti-β2GPI/β2GPI complex.(4) The expression of TLR4was significantly increased with stimulation of anti-β2GPI/β2GPI complex. EGCG and fluvastatin reduced anti-β2GPI/β2GPI complex-enhanced TLR4mRNA and its protein levels.(5) The phosphorylation of NF-κB and MAPKs in THP-1cells were also significantly increased with stimulation of anti-β2GPI/β2GPI complex, in a manner of time-dependence. EGCG and fluvastatin dose-dependently decrease phosphorylation of NF-κB and MAPKs in the presence of anti-β2GPI/β2GPI (p<0.05).Conclusions:(1) The TF and TNF-a expression in THP-1cells can be significantly up-regulated with treatment of anti-β2GPI/β2GPI complex.(2) EGCG and fluvastatin can decrease anti-β2GPI/β2GPI-induced TF and TNF-a expression in THP-1cells.(3) EGCG and fluvastatin can reduce the expression of TLR4, phosphorylation of NF-κB and MAPKs in the presence of anti-β2GPI/β2GPI complex.(4) EGCG and fluvastatin attenuate anti-β2GPI/β2GPI-induced activation in THP-1cells through inhibiting the intracellular signal transduction pathway of TLR4-MAPKs-NF-κB axis and may serve as preventive and therapeutic agents for APS.更多还原