【作者】 林伊凤；

【导师】 张农； 马端； 赵仲华；

【作者基本信息】 复旦大学 ， 病理学， 2008， 博士

【摘要】 前言肾小球硬化是各种原因所致肾小球损伤和病变的共同结局,其重要的病理特征之一是肾小球内细胞外基质（extracellular matrix,ECM）的异常沉积。ECM合成/降解失衡以及ECM代谢调控的异常是导致肾小球硬化发生的重要机制。肾小球系膜细胞（mesangial cell,MsC）在肾小球内ECM代谢及调控过程中起重要作用。因此,加速和调控肾小球系膜细胞的凋亡以及探讨其与肾小球硬化发生、发展的相互关系是近年来对系膜增生型肾小球肾炎的研究热点之一。肾小球系膜细胞作为肾小球内功能活跃的固有细胞之一,其增生和凋亡失衡在肾小球损伤和肾小球硬化的发生发展过程中具有关键性作用。体内外大量研究表明,反复或持续性肾小球损伤导致的持续性MsC增生能加速并且导致系膜区ECM沉积增加,引起不可逆性肾小球损伤,终致肾功能丧失。近来研究结果提示,抑制MsC增殖,促进MsC凋亡可以限制系膜增生型肾小球肾炎的发展。肾小球内MsC异常增生受到抑制后,Ⅳ型胶原、Laminin及Fibronectin明显减少,系膜区ECM沉积亦减少,从而限制了疾病的发展。组织因子途径抑制物（tissue factor pathway inhibitor,TFPI）是体内主要的生理性抗凝物质之一。TFPI能特异性地与FXa以及FVIIa/TF复合物结合,形成TFPI-FXa-FVIIa-TF四元复合物,灭活FVIIa-TF复合物的活性,发挥负反馈性抑制外源性凝血途径的作用。除了其主要的抗凝作用外,TFPI还具有抗炎、抗血管生成、抗增殖以及诱导细胞凋亡等生理功能。体外研究结果提示,TFPI能抑制培养的人主动脉平滑肌细胞增殖,并且也能诱导人脐静脉内皮细胞发生凋亡。同时国外研究发现,培养的人MsC能分泌TFPI,并且能有效地抑制TF的活性,从而抑制纤维蛋白的形成。TFPI对于治疗肾小球肾炎也具有重要意义。临床研究表明,在人新月体型肾小球肾炎中TFPI表达明显上升,因此推测它在治疗纤维蛋白依赖性肾小球肾炎中有重要意义。本课题采用制备大鼠抗Thy-1肾炎模型、细胞培养、免疫组织化学、免疫细胞荧光、Western blot、基因转染、PCR等实验方法,分别从体内、体外两方面观察TFPI在肾小球肾炎发展过程中的表达;观察rTFPI对大鼠MsC凋亡的影响,并初步探讨这种作用所涉及的信号通路,以期能探讨和初步明确rTFPI在肾小球肾炎、肾小球硬化等病理过程中的作用及意义,并为系膜增生型肾小球肾炎临床治疗提供新的思路和实验依据。第一部分TFPI的基因表达和产物纯化目的获得高纯度的重组TFPI（recombinant TFPI,rTFPI）。方法扩增TFPI基因,构建大肠杆菌表达质粒pET-28a（+）-TFPI,在大肠杆菌BL21（DE3）中诱导表达重组TFPI,用离子交换层析法纯化重组TFPI,Western blot对纯化产物进行鉴定,稀释凝血酶原时间法检测重组TFPI的抗凝活性。结果大肠杆菌BL21（DE3）表达重组TFPI,后者以包涵体形式存在于菌体内,dPT实验结果,与对照组相比,重组TFPI PT延长16.2s。小结大肠杆菌BL21（DE3）成功表达重组TFPI,经复性、纯化后重组TFPI纯度大于95%,并具有较高的抗凝活性。第二部分TFPI在大鼠抗Thy-1肾炎模型及人肾小球肾炎中的表达及其对肾小球MsC凋亡的影响目的探讨TFPI在大鼠抗Thy-1系膜增生型肾小球肾炎模型以及人肾小球轻微病变和系膜增生型肾小球肾炎组织肾小球中的表达;明确TFPI对肾小球MsC凋亡的影响;寻找TFPI蛋白中诱导MsC凋亡的功能区。方法制备兔抗大鼠Thy-1抗血清,并利用该抗血清复制大鼠抗Thy-1肾小球肾炎模型,收集人肾小球轻微病变和系膜增生型肾小球肾炎肾穿活检标本,采用免疫组织化学结合图像分析的方法,观察内源性TFPI的表达。外源性加入rTFPI、rTFPI_1-161（包含TFPI N末端和第一、二结构域KD1、KD2）、MBP-TFPI_162-276（包含TFPI第三结构域KD3和C末端）、MBP-TFPI KD3、MBP-TFPI C末端刺激培养的大鼠肾MsC,采用Hoechst 33258染色、流式细胞分析技术、DNA片段凝胶电泳（DNA Ladder）以及Western blot等方法分析MsC凋亡情况。结果大鼠抗Thy-1肾小球肾炎模型中,肾小球内TFPI在肾炎第14天显著表达,21天表达更明显,28天到达高峰。与人肾小球轻微病变肾穿组织相比,系膜增生型肾小球肾炎组织肾小球内TFPI的表达明显增高。经外源性rTFPI刺激后,凋亡的MsC数量明显增加,并呈现时间和剂量依赖性,分别为对照组的2.1、3.0、4.9倍,rTFPI作用组出现明显的阶梯状DNA条带,活性形式的Caspase-3蛋白表达也明显高于对照组,并呈剂量依赖性表达。外源性加入rTFPI、rTFPI_1-161、MBP-TFPI_162-276、MBP-TFPIKD3、MBP-TFPI C末端后,rTFPI组、MBP-TFPI_162-276组、MBP-TFPI C末端组可见明显的MsC凋亡现象,而rTFPI_1-161组、MBP-TFPI KD3组无明显MsC凋亡。小结大鼠抗Thy-1肾小球肾炎模型中,肾小球内TFPI于炎症后期开始表达,并随着炎症时间的延长而表达增加;人系膜增生型肾小球肾炎中肾小球内TFPI的表达高于肾小球轻微病变者;外源性rTFPI能诱导大鼠MsC发生凋亡,并呈现时间和剂量依赖性,PDGF刺激MsC增殖后rTFPI仍具有该作用;rTFPI诱导MsC发生凋亡的功能区位于其C末端。第三部分rTFPI诱导MsC凋亡的相关信号通路的初步研究目的探讨rTFPI诱导MsC凋亡是否与组织因子（tissue factor,TF）有关;对rTFPI诱导MsC凋亡所涉及的信号通路进行初步研究。方法采用免疫细胞荧光的方法通过激光共聚焦观察rTFPI诱导MsC凋亡是否是通过与细胞膜上的TF结合来完成的;采用Western blot的方法检测rTFPI刺激MsC发生凋亡后,线粒体信号通路蛋白Bel-2、死亡受体Fas以及PI3K-Akt信号通路的表达改变;用Fas中和抗体阻断Fas通路后,采用Hoechst 33258染色观察MsC凋亡变化;采用RT-PCR、酶切、连接方法,构建真核表达质粒pEGFPcl-Akt1,将其稳定转染至MsC中,筛选出Akt1高表达克隆,用外源性rTFPI分别刺激正常MsC、转染空载pEGFPcl的MsC以及转染pEGFPcl-Akt1的MsC,采用Hoechst 33258染色实验和流式细胞分析技术观察三组MsC凋亡情况。结果外源性rTFPI刺激MsC后进行免疫荧光检测,rTFPI以红色荧光标记,MsC细胞膜上TF以绿色荧光标记,两者未叠加;外源性rTFPI刺激MsC凋亡后,线粒体信号通路蛋白Bcl-2表达不变,死亡受体Fas表达增加,并呈现剂量依赖性,加入Fas中和抗体阻断死亡受体Fas通路后,rTFPI诱导的MsC凋亡作用减弱;Western blot结果显示,rTFPI刺激MsC发生凋亡后,磷酸化Akt、磷酸化IκB表达均明显减弱;构建pEGFPcl-Akt1真核表达质粒,经稳定转染MsC后获得Akt1高表达克隆。将外源性rTFPI分别刺激正常MsC、转染空载pEGFPcl的MsC以及转染pEGFPcl-Akt1的MsC,Hoechst 33258染色实验和流式细胞分析结果显示,正常MsC组和转染空载pEGFPcl-MsC组细胞凋亡明显,而转染pEGFPcl-Akt1-MsC组细胞凋亡被明显抑制。小结外源性rTFPI诱导MsC凋亡与TF起始的信号转导途径无关。线粒体信号通路蛋白Bcl-2不参与rTFPI诱导的MsC凋亡,而死亡受体Fas/FasL信号转导通路参与该过程,阻断该通路后,rTFPI诱导的MsC凋亡作用可被明显抑制;rTFPI诱导MsC凋亡后,PI3K/Akt信号转导通路中的Akt和IκB磷酸化明显被抑制,上调MsC中Akt1表达可拮抗rTFPI引起的MsC凋亡,提示PI3K/Akt信号转导通路也是参与rTFPI诱导MsC凋亡的主要信号转导途径之一。结论1.MsC可表达和分泌TFPI;在大鼠抗Thy-1肾炎模型标本中,肾小球内TFPI表达于炎症后期并随炎症时间延长而表达增强,在人肾穿标本中,系膜增生型肾小球肾炎组织肾小球中TFPI的表达明显高于肾小球轻微病变者,提示TFPI可能参与了系膜增生型肾小球肾炎的病理过程。2.rTFPI可以诱导培养的大鼠MsC发生凋亡,并具有时间和剂量依赖性,即使是在MsC大量增殖的环境中,仍具有该作用;rTFPI诱导MsC发生凋亡的功能区主要位于其C末端。3.rTFPI诱导MsC凋亡与TF起始的信号转导通路无关。在rTFPI诱导MsC凋亡的过程中,死亡受体Fas/FasL信号转导通路被激活,而介导细胞增殖的PI3K/Akt信号通路活性受到抑制,提示死亡受体Fas/FasL信号转导通路和PI3K/Akt信号通路均是参与rTFPI诱导MsC凋亡的主要信号通路。更多还原

【Abstract】 IntroductionGlomerulosclerosis is a final common outcome of glomerular injury in various types of human glomerular diseases,which is characterized with the proliferation of glomerular mesangial cell（MsC） and abnormal accumulation of extracellular matrix（ECM） in glomeruli.There is increasing evidence that the imbalance of ECM synthesis and degradation may be an important mechanism of glomerulosclerosis.Glomerular MsC plays an important role in the regulation of ECM metabolism.Recent research points are focused on the regulation of MsC apoptosis and its correlation with the development of glomerulosclerosis.As resident cells in glomerulus,mesangial cells play an important role not only in maintaining the normal structure and functions of glomerulus,but also in acute glomerulonephritis and progressive glomerulosclerosis.In many glomerular sclerosing diseases,mesangial cell proliferation is a major important pathological feature in progressive glomerular injuries.Apoptosis is the major cell clearance mechanism for removal of large numbers of excess mesangial cells in experimental mesangial proliferation. Recent researches have demonstrated that apoptosis may be limited increases in MsC number,to mediate removal of surplus MsCs and to resume the structure and function of glomerular.Tissue factor pathway inhibitor（TFPI） is a Kunitz-type serine protease inhibitor which exhibits a strong and specific inhibitory activity against the tissue factor（TF）-mediated initiation of the blood coagulation cascade.The function of TFPI as the physiologic inhibitor of TF-initiated coagulation has promoted interests in applying recombinant TFPI （rTFPI） as a substitution therapy in some inflammations and cancers.Growing evidence indicates that TFPI has antiangiogenic and antitumor activities and may play a role in cell anti-proliferation and apoptosis.Some studies show that human recombinant TFPI（hrTFPI） prevented the proliferation of cultured human neonatal aortic smooth muscle cells and induced apoptosis in cultured human endothelial cells.In addition to endothelial cells, human MsCs also have the ability to produce TFPI which inhibits fibrin formation.Some animal tests and clinical trials also show that TFPI is strongly expressed in crescentic glomerulonephritis and has the potential to be of therapeutic benefit in the management of fibrin dependent human glomerulonephritis.This suggested us TFPI may has the function in the therapeusis of glomerulonephritis.The aim of the present study is to observe the expression of TFPI in the progression of mesangial proliferative glomerulonephritis;to investigate the effect of rTFPI on the apoptosis of rat cultured MsCs and find the functional domain of rTFPI which leads to apoptosis of MsC and to analyze the possible signaling pathways involved in rTFPI-induced apoptosis in MsC.The purpose of our research was to elucidate the function and significance of rTFPI in mesangial proliferative glomerulonephritis and provide new sight and experiments data for the therapeusis of these glomerulonephritis.PartⅠExpression and purification of recombinant tissue factor pathway inhibitorObjective To obtain the purified recombinant TFPI.Methods pET-28a（+） and TFPI gene was treated with the restriction endonucleases of NcoI and NotI.The recombinant plasmid of pET-28a（+）-TFPI was transformed to E.coli. rTFPI was purified by ion exchange chromatography and characterized by Western blot. Dilute-prothrombin time（dPT） was used to examined the anti-coagulation activity of rTFPI.Results Recombinant TFPI（rTFPI） was expressed successfully in E.coli as inclusion bodies.Compared with control,rTFPI prolonged prothrombin time for 16.2s.Conclusions After refolding and purification,recombinant TFPI was successfully obtained with purity of more than 95%with highly anti-coagulation activity.PartⅡExpression of TFPI in rat anti-Thy-1 nephritis model and human glomerulonephritis and its effect on MsC apoptosisObjective To explore the expression of TFPI in glomerulus in rat anti-Thy-1 glomerulonephritis model and human glomerulonephritis,determinate the effect of rTFPI on apoptosis in cultured rat MsC and find the functional domain of rTFPI which leads to MsC apoptosis.Methods Rabbit anti-rat Thy-1 serum was used to construct rat anti-Thy-1 glomerulonephritis model.Immunohistochemistry and image analysis software were used to analyze the expression of TFPI in glomerulus in rat anti Thy-1 glomerulonephritis model and human glomerulonephritis with different pathologic types.Hoechst 33258、flow cytometry、DNA fragmentation and Western blot were used to analyze apoptosis of MsC which stimulated by rTFPI,rTFPI_1-161.MBP-TFPI_162-276,MBP-TFPI KD3 and MBP-TFPI C terminal,respectively.Results In rat anti-Thy-1 glomerulonephritis,TFPI was begun to express obviously in the glomeruli at day 14,markedly increased at day 21 and peaked at day 28.Compared with glomerular minor lesion,TFPI in glomerulus was highly expressed in human mesangial proliferative glomerulonephritis.Apoptotic MsCs were increased after stimulated by rTFPI in a dose-and time-dependent manner.The characteristic pattern of fragmentation of DNA in multiple of 180-200bp was found after MsCs were treated with rTFPI,rTFPI has an effect on MsC apoptosis when MsC is proliferating induced by PDGF.The expression of active Caspase-3 was also increased in a dose-dependent manner after added rTFPI.There was no apoptotic MsCs after treated with rTFPI_1-161 and MBP-TFPI KD3,whereas typical images of apoptotic MsCs were found in which stimulated by rTFPI,MBP-TFPI_162-276 and MBP-TFPI C terminal.Conclusions In rat anti-Thy-1 glomerulonephritis,TFPI in glomerulus was expressed in the late period and increased with the progress of nephritis.TFPI was expressed higher in glomerulus in human mesangial proliferative glomerulonephritis than that in glomerular minor lesion,rTFPI induced apoptosis in cultured rat MsC in a dose-and time-dependent manner by its C terminal even though MsC was proliferating.PartⅢStudy of the related signaling pathway on rTFPI-induced apoptosis in cultured rat MsCObjective To explore whether rTFPI induced MsC apoptosis via binding to TF and the possible signaling pathway involved in rTFPI-induced MsC apoptosis.Methods Immunofluorescence was used to determine whether rTFPI induced MsC apoptosis via binding to TF.Western blot was used to analyze the expressions of Bcl-2,Fas, phospho-Akt,total Akt,phospho-IκB.Hoechst 33258 was used to determine apoptosis after blocked the Fas pathway by its neutralizing antibody.RT-PCR,restriction endonucleases digestion and ligation were used to reconstructing eukaryotic expression plasmid pEGFPc1-Akt1.Lipofectamine^TM 2000 was used to stably transfect Akt1 vectors into MsC. Western blot analysis were performed to verify the transfection.Hoechst 33258 and flow cytometry were used to analyze apoptosis in normal MsC,pEGFPcl-MsC and pEGFPc1-Akt1-MsC treated by rTFPI.Results The red fluorescence which represented rTFPI didn’t merge in the green fluorescence represented TF in MsC cytomembrane.Treated with rTFPI in MsC,there were no changes in the Bcl-2 protein expression,but the expression of Fas was increased in a dose-dependent manner.After blocked Fas signalling pathway by its neutralizing antibody, apoptotic MsCs induced by rTFPI were decreased obviously.By contraries,phospho-Akt and phospho-IκB,which represented the activation of PI3K/Akt pathway,were decreased in a dose-dependent manner after treated with rTFPI.After over-expressed Akt1 gene, apoptosis was prevented in MsC which were transfected with Akt1 although treatment with rTFPI.However,the normal MsCs and MsCs which were transfected pEGFPc1 were undergone apoptosis after rTFPI treatment.Conclusions rTFPI-induced apoptosis of MsCs is not via binding to TF.Bcl-2 did not involve in the signaling pathway which induces apoptosis in MsCs by rTFPI stimulation. Death receptor Fas/FasL signalling pathway involved in the progress of apoptosis in MsC induced by rTFPI.Blocked this pathway,apoptotic MsCs were decreased obviously. PI3K/Akt signalling pathway was one of the significant pathways which mediated rTFPI-induced MsC apoptosis.Up-regulated Akt was able to prevent the apoptosis progress.Conclusions1.MsC is one kind of cells which expresses and secretes TFPI.In rat anti-Thy-1 glomerulonephritis,TFPI in glomerulus was expressed in the late period and increased with the progress of nephritis.TFPI was expressed higher in glomerulus in human mesangial proliferative glomerulonephritis than that in glomerular minor lesion.This suggests that TFPI may involve in the pathological progress of mesangial proliferative glomerulonephritis.2.rTFPI induced apoptosis in cultured rat MsC in a dose- and time-dependent manner by its C terminal. 3.rTFPI-induced apoptosis of MsCs is not via binding to TF.Bcl-2 did not involve in the signalling pathway which induces apoptosis in MsCs by rTFPI stimulation.Death receptor Fas/FasL signalling pathway involved in the progress of apoptosis in MsC triggered by rTFPI.PI3K/Akt signalling pathway was one of the significant pathways which mediated rTFPI-induced MsC apoptosis.更多还原