【作者】 刘心利；

【导师】 朱付凡；

【作者基本信息】 中南大学 ， 妇产科学， 2012， 博士

【摘要】 抗磷脂综合征(Antiphospholipid syndrome, APS)是一种系统性自身免疫性疾病,血清抗磷脂抗体(antiphospholipid antibody, aPL)阳性和血栓前状态为APS的两大重要血清学特征,这种血清学特征与栓塞的发生和病理妊娠密切相关。APS发病机制不明,血栓形成一直以来被认为是APS发病的重要因素,而内皮细胞活化则是血栓形成的重要原因。抗体浓度及感染与APS的发病及预后关系密切,国内外学者普遍认为中高浓度情况下,浓度越高、病情越重、预后越差,感染则可加重APS病情。导致此种现象的机制未明,内皮细胞是否参与其中,国内未见此方面研究。aPL活化内皮细胞具有特定的信号传导通路,并非简单的磷脂结合途径。大部分aPL直接与β2糖蛋白I (β2GPI)结合,aPL/β2GPI复合物进一步结合于细胞表面。目前细胞的Toll样受体(TLR)信号传导通路尤其是TLR2和TLR4信号通路是研究的热点,国外研究发现TLR2参与aPL活化内皮细胞的信号传导中,而TLR4是否参与其中存在争议,国内未见相关研究。内皮细胞活化后可分泌多种细胞因子,本文选择E-选择素(SELE)、单核细胞趋化蛋白1(MCP-1)、白细胞介素6(IL-6)和白细胞介素8(IL-8)四种常用且敏感的指标用以监测内皮细胞活化后的分泌功能,其中E-选择素mRNA表达量增加或降低时,其蛋白的表达水平发生相应的上调或下调,因此本文分别从mRNA及蛋白水平对E-选择素的表达进行检测,若二者的表达均增加或减少,则从一方面说明了实验结果正确性。本文所采用的aPL为从APS患者血清中提纯的IgG,为排除正常IgG对实验的影响,选择正常人群血清中提纯的IgG作为正常对照。本文拟分为三个部分,第一部分体外培养脐静脉内皮细胞(human umbilical vein endothelial cell, HUVEC),分别用正常对照IgG及不同浓度APS患者IgG刺激细胞,探讨不同浓度aPL对内皮细胞分泌E-选择素、IL-6、IL-8、MCP-1的影响。第二部分探讨TNF-α干预下内皮细胞TLR2/4表达情况其TNF-α干预下aPL对血管内皮细胞分泌E-选择素、IL-6、IL-8、MCP-1的影响,进一步研究APS血管内皮细胞活化的机制,并为治疗APS提供新靶点。第三部分通过研究卡介菌多糖核酸(BCG-PSN)对aPL刺激内皮细胞分泌E-选择素、IL-6、IL-8、 MCP-1的影响,探讨BCG-PSN是否有可能成为治疗APS的新选择药物。第一章不同浓度抗磷脂抗体对内皮细胞分泌E-选择素、IL-6、IL-8. MCP-1影响的研究目的：观察不同浓度抗磷脂抗体对血管内皮细胞分泌功能的影响。方法：体外培养人脐静脉内皮细胞,分别用PBS(空白组)、正常人血清提取的IgG(正常对照组)和不同浓度APS患者血清提取的IgG (APS IgG)(5%、10%和20%)干预细胞。采用实时荧光定量PCR及流式细胞学技术,检测细胞E-选择素的表达,采用酶联免疫吸附试验测定细胞分泌MCP-1、IL-6和IL-8的表达,采用Western blot检测内皮细胞TLR2和TLR4的表达。结果：正常对照组内皮细胞SELE、MCP-1、IL-6和IL-8表达量与空白组比较,无统计学差异(p>0.05); APSIgG可诱导内皮细胞SELE. MCP-1、IL-6和IL-8表达增加,与空白组、正常对照组比较及不同浓度APS IgG组之间两两比较,差异有显著统计学意义(p<0.01)；空白组、正常对照组及5%APS IgG组两两比较,内皮细胞TLR2表达量无明显差异(p>0.05),10%及20%APS IgG组TLR2表达量与空白组及正常对照组比较及两组之间比较,差异有统计学意义(p<0.05)；各组之间TLR4表达量无明显差异(p>0.05)。结论：抗磷脂抗体刺激内皮细胞分泌E-选择素、IL-6、IL-8、 MCP-1增加且作用具有浓度依赖性,浓度越高,作用越强。第二章TNF-a对内皮细胞TLR2/4表达的影响及APS发病机制的研究目的：探讨TNF-a干预下aPL刺激内皮细胞分泌E-选择素、IL-6、IL-8、 MCP-1的情况及TLR2和TLR4在抗磷脂抗体刺激内皮细胞分泌E-选择素、IL-6、IL-8、MCP-1中的作用。方法：(1)一半细胞采用肿瘤坏死因子a (TNF-a)预刺激24小时后去除刺激因素8小时,一半细胞未经TNF-a预刺激但余处理相同,收集一部分TNF-a预刺激及未预刺激细胞,采用Western blot检测TNF-a预处理对内皮细胞TLR2和TLR4表达的影响,余下的TNF-a预刺激及未预刺激细胞分别用脂磷壁酸(LTA)、正常对照IgG及不同浓度APS IgG(5%、10%和20%)进行干预,采用实时荧光定量PCR及流式细胞学技术,检测细胞E-选择素的表达,采用酶联免疫吸附试验测定细胞分泌MCP-1、IL-6和IL-8的表达；(2)分别加入抗-TLR2或(和)抗-TLR4阻断抗体阻断TLR2或(和)TLR4信号传导通路后,用20%浓度APS IgG干预细胞,采用实时荧光定量PCR及流式细胞学技术,检测细胞E-选择素的表达,采用酶联免疫吸附试验测定细胞分泌MCP-1、IL-6和IL-8的表达。结果：经TNF-a预刺激后,细胞TLR2表达量较未经TNF-a预刺激细胞明显增加,差异有统计学意义(p<0.01),TLR4表达量无明显变化(p>0.05)；经TNF-a预处理及未预处理细胞均用TLR2特异性激动剂LTA进行干预,经TNF-a预处理组SELE、MCP-1、IL-6和IL-8表达量较未经TNF-a预处理组明显增高,差异有统计学意义(p<0.01)：经TNF-a预刺激后再用同浓度APS IgG干预细胞,SELE、 MCP-1、IL-6和IL-8表达量与仅用APS IgG干预组比较明显增高,差异有统计学意义(p<0.01),不同浓度APS IgG组与正常对照组四组之间两两比较,差异均有统计学意义(p<0.01)；抗-TLR2+APS IgG组及抗-TLR2+抗-TLR4组内皮细胞SELE、 MCP-1、IL-6和IL-8表达量,较APS IgG组明显降低,差异有统计学意义(p<0.01)；抗-TLR4+APS IgG组内皮细胞SELE. MCP-1、 IL-6和IL-8表达量,与APS IgG组比较无明显改变,差异无统计学意义(p>0.05)。结论：(1)TNF-a干预下抗磷脂抗体刺激内皮细胞分泌E-选择素、IL-6、 IL-8、MCP-1的作用增强。(2)抗磷脂抗体通过TLR2信号传导通路刺激内皮细胞使其E-选择素、IL-6、IL-8、MCP-1分泌增加。第三章卡介菌多糖核酸对抗磷脂抗体刺激内皮细胞分泌E-选择素、IL-6、IL-8、MCP-1的影响目的：探讨卡介菌多糖核酸(BCG-PSN)干预下抗磷脂抗体刺激内皮细胞分泌E-选择素、IL-6、IL-8、MCP-1的情况。方法：用APS患者血清提取的IgG处理内皮细胞,APS IgG处理前预先加入不同浓度BCG-PSN(0、25、50和100μg/ml),采用实时荧光定量PCR及流式细胞学技术,检测细胞E-选择素表达,采用ELISA测定细胞分泌MCP-1、IL-6和IL-8的表达。结果：BCG-PSN干预下APS IgG所诱导的内皮细胞SELE.MCP-1、IL-6和IL-8表达明显降低,APS IgG组及不同浓度BCG-PSN组之间两两比较,差异均有统计学意义(p<0.01)。结论：卡介菌多糖核酸干预下抗磷脂抗体刺激内皮细胞分泌E-选择素、IL-6、IL-8、MCP-1的作用减弱,提示它可以用于APS的治疗。更多还原

【Abstract】 Antiphospholipid syndrome (APS) is a systemic autoimmune disease, the presence of antiphospholipid antibody (aPL) in the serum and generalized prothrombotic state are the two major characteristics, and this serological characteristics are closely related to embolism and pathological pregnancy. The pathogenesis of APS is still unclear, prothromboticstate is one of the important factors, and the important factor required for the formation of prothromboticstate is endothelial cells’damage and activation. About the relation between the concentration of aPL, infection and the incidence and prognosis of APS, some foreign scholars believe that the higher the concentration, the worse the prognosis, infection can make APS worse. What makes this phenomenon? Is the activation of endothelial cell involved in it? No such research has been seen in China.One of the major receptors for aPL is β2-glycoprotein I (β2GPI). aPL/β2GPI complex further binds to annexin A2, then combines with phospholipids on the cell membrane, however, annexin A2does not have the transmembrane structure, cell signal cannot be conducted. Many researches have been focused on toll-like receptor (TLR) signaling pathway and found that TLR2mediated the signal transduction, disagreement was existed about the role of TLR4, no such research in China. After activation, the synthesis and secretion of adhesion molecule such as E-selectin (SELE) significantly increased, a number of inflammatory cytokines such as interleukin-1β(IL-1β), interleukin-6(IL-6),interleukin-8(IL-8), tumor necrosis factor-a (TNF-a), monocyte chemoattractant protein-1(MCP-1) also increased significantly, resulting in a proinflammatory and procoagulant state. Among them, E-selectin、 IL-6、IL-8、MCP-1are the most sensitive and frequently used factors in monitoring the endothelial cells’activation extent. The consistent change of SELE mRNA and pretein levels means the correctness of the experimental results. Limited by the experimental techniques and conditions, the aPL used in our research is the purified IgG from APS patients, the purified IgG from normal person was used as normal control to exclude the possible effects of the normal types of IgG.The paper is divided into three parts:the first part was to investigate the effects of various titers of aPL on endothelial cells. Culturing human umbilical vein endothelial cells (HUVECs) in vitro, stimulating them with purified IgG proteins from APS or normal non-pregnant women. Endothelial cell secretion function was assessed by measuring mRNA and protein levels of E-selectin by the means of real-time fluorescent quantitative-polymerase chain reaction (RT-PCR) and flow cytometry (FACS), the protein levels of MCP-1, IL-6and IL-8were measured by mean of enzyme-linked immunosorbent assay (ELISA), TLR2and TLR4 were measured by Western blot. The second part was to study the effect of TNF-α and the role of TLR2and TLR4in activation of the endothelial cells by aPL. By blocking TLR2or (and) TLR4signal transduction way, we study the role of TLR2/4directly. The third part was about investigating the effects of BCG polysaccharides nucleic acid (BCG-PSN) in activation of endothelial cells by aPL, to explore whether BCG-PSN can be used in APS. Chapter1Secretion of E-selectin, IL-6, IL-8and MCP-1by endothelial cell stimulated by various titers of antiphospholipid antibodyObjective:To study the expression of E-selectin, IL-6, IL-8, MCP-1on endothelial cell when stimulated by various titers of aPLs.Methods:HUVECs were cultured in vitro, and stimulated with aPLs from APS (5%,10%,20%) or normal non-pregnant women. Endothelial cell activation was assessed by measuring mRNA and protein levels of E-selectin by the means of RT-PCR and FACS, the protein levels of MCP-1, IL-6and IL-8were measured by mean of ELISA, Western blot analysis was used to measure the protein levels of TLR2and TLR4.Results:Stimulated by aPL showed a significant increase in SELE, IL-6, IL-8, MCP-1expression, comparing every two groups of blank, normal control or aPL (5%,10%,20%) groups, p<0.01.There are no significant difference on TLR2expression between the groups of blank, normal and5%aPL(p>0.05), TLR2expression in10%and20%aPL groups were significantly increased compared with the blank and normal control groups or with each other (p<0.05). aPL has no effect on the expression of TLR4(p>0.05)Conclusions:Expression of E-selectin, IL-6, IL-8and MCP-1on endothelial cell increased when stimulated by aPL, the higher the antibody concentration, the higher the expression. Chapter2The role of TNF-a, TLR2and TLR4in activation of endothelial cell by antiphospholipid antibodiyObjective:To investigate the role of TNF-a, TLR2/4in secretion function of endothelial cell when stimulated by antiphospholipid antibodiy.Methods:(1) HUVECs were cultured in vitro, part of them were stimulated with100ng/ml TNF-a for24hours followed by an8h wash-out, collecting some TNF-pretreated and non-pretreated cells, using Western blot analysis to measure the protein levels of TLR2and TLR4. The rest cells were stimulated with LTA or aPLs from APS women. Endothelial cell activation was assessed by measuring mRNA and protein levels of E-selectin by the means of RT-PCR and FACS, the protein levels of MCP-1, IL-6and IL-8were measured by mean of ELISA;(2) HUVECs were cultured in vitro and stimulated with100ng/ml TNF-a for24hours followed by an8h wash-out, then treated with aPL, Anti-TLR2antibodies or/and anti-TLR4antibodies were used before adding aPL to block TLR2or/and TLR4signal conduct pathways. Endothelial cell activation was assessed by measuring mRNA and protein levels of E-selectin by the means of RT-PCR and FACS, the protein levels of MCP-1, IL-6and IL-8 were measured by mean of ELISA.Results:After pre-stimulation by TNF-a, the TLR2expression on HUVEC was significantly increased compared with the non-pretreated group (p<0.01), the TLR4expression had no change (p>0.05). stimulated by LTA, the specific agonist of TLR2, the expression of SELE, IL-6, IL-8, MCP-1were significantly increased in TNF-apretreated group compared with the non-pretreated group (p<0.01). Under the same concentration of aPL, the expression of SELE, IL-6, IL-8, MCP-1in TNF-pretreated group were significantly higher than non-pretreated group (p<0.01).Blocking TLR2signal conduct pathways significantly attenuated the expression of SELE, IL-6, IL-8, MCP-1expression on HUVEC stimulated by aPL (p<0.01). blocking TLR4did not have significant effect on the expression of SELE, IL-6, IL-8, MCP-1stimulated by aPL HUVEC (p>0.05).Conclusions:(1) TNF-apre-treated endothelial cell secretes more SELE, IL-6, IL-8, MCP-1than non-pre-treated cell when stimulated by aPL.(2) Antiphospholipid antibodies activate endothelial cell via TLR2signal transduction pathways. Chapter3The impact of BCG-polysaccharides nucleic acid on E-selectin, IL-6, IL-8and MCP-1expression on endothelial cell stimulated by antiphospholipid antibodyObjective:To investigate the impact of BCG-polysaccharides nucleic acid (BCG-PSN) on secretion function of endothelial cell stimulated by antiphospholipid antibody.Methods:HUVECs were cultured in vitro, and stimulated with100ng/ml TNF-a for24hours followed by an8h wash-out, then treated with20%of APS aPLs, APS aPLs treated groups were also treated with different concentrations of BCG-PSN (0,25,50and100μg/ml). Endothelial cell activation was assessed by measuring mRNA and protein levels of E-selectin by the means of RT-PCR and FACS, the protein levels of MCP-1, IL-6and IL-8were measured by mean of ELISA.Results:APS IgG induced endothelial cells’ SELE, IL-6, IL-8, MCP-1expression increasing, while the intervention of BCG-PSN attenuated APS IgG-induced SELE, IL-6, IL-8, MCP-1expression, compared any two of BCG-PSN groups (0,25,50and100μg/ml), the differences were statistically significant (p<0.01). Conclusion:Antiphospholipid antibody induced secretion of E-selection, IL-6, IL-8, MCP-1on endothelial cell decreased when interfered by BCG-polysaccharides nucleic acid, suggesting that it may be useful for the treatment of APS.更多还原