Toll样受体3通过内外源性途径诱导细胞凋亡的分子机制

发布时间：2018-03-18 21:52

  本文选题：TLR3　切入点：Poly　出处：《中南大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的:Toll样受体(Toll-like receptors,TLRs)是病原相关分子模式识别受体,与相应配体结合后激活其下游信号转导途径,诱导炎症反应和天然免疫应答,同时促进抗原提呈细胞的活化并介导获得性免疫反应,从而在宿主抗微生物感染中发挥重要作用。TLR3是病毒双链RNA识别受体,活化后能激活NF-κB和IRF3途径来抵抗病毒感染。此外,TLR3活化后还能诱导细胞损伤,并破坏体内血管屏障引起病毒扩散,从而加速疾病的进程。本研究的目的是通过分析TLR3损伤的关键信号分子,尝试抑制此关键分子来干预TLR3诱导的细胞损伤,为临床选择相关靶点延缓感染性疾病的进程提供理论基础。 方法:1、RT-PCR检测基因的表达。2、流式细胞术检测蛋白质的表达。3、Western blot检测信号转导分子的活化。4、PI/Annexin V染色检测细胞凋亡水平。5、抗体中和实验验证相关信号分子与细胞凋亡的相关性。6、构建真核表达载体pcDNA3.1+/ΔNp63α并转染内皮细胞,检测ΔNp63α对TLR3诱导细胞凋亡的影响。 结果:RT-PCR结果显示人脐静脉内皮细胞HUVEC较高表达Toll样受体家族中的TLR2、3、4和5,低表达TLR6和9,不表达TLR1、7、8和10。作为对照人外周血单个核细胞表达所有TLR1-10基因。利用TLR3配体dsRNA的类似物PolyⅠ:C刺激HUVEC,结果显示PolyⅠ:C上调TLR3基因表达并呈剂量依赖性,FACS分析结果显示HUVEC细胞表达TLR3蛋白质。Western blot检测发现PolyⅠ:C作用TLR3诱导了其下游信号分子NF-κB的活化,并剂量依赖性地上调细胞因子IL-1β、IL-6、IL-8、TNF-α、IFN-β的基因表达,表明HUVEC表达功能性的TLR3受体。此外,PolyⅠ:C处理的HUVEC细胞表现出与Staurosprine(一种凋亡诱导化学物质)处理后相似的形态学改变,PI/Annexin V染色显示PolyⅠ:C诱导了剂量相关的细胞凋亡;这种作用由TLR3所介导,因为TLR3的中和抗体能显著抑制PolyⅠ:C所诱导的细胞凋亡。PolyⅠ:C处理的HUVEC同时活化了caspase 8和9及其下游分子PARP,caspase和PARP抑制剂能显著抑制PolyⅠ:C诱导的凋亡,表明TLR3通过内源性途径(又称线粒体途径)和外源性途径(又称死亡受体途径)诱导细胞凋亡。由于我们同期的研究还发现TLR3诱导的细胞凋亡受p53家族成员TAp63α的调控,因此我们构建了TAp63α的显性负性异构体ΔNp63α的真核表达载体,转染HUVEC后显示其能显著抑制PolyⅠ:C诱导的细胞凋亡。 结论:HUVEC表达功能性的TLR3,活化的TLR3通过内、外源性两条途径诱导HUVEC细胞凋亡。TAp63α的显性负性突变异构体ΔNp63α降低PolyⅠ:C诱导的细胞凋亡,推测TAp63α在TLR3诱导的细胞凋亡中发挥重要作用。
[Abstract]:Objective: Toll-like receptor (TLRs) is a pathogen-associated molecular pattern recognition receptor (TLRs), which binds to the corresponding ligand and activates its downstream signal transduction pathway and induces inflammatory response and innate immune response. At the same time, it promotes the activation of antigen-presenting cells and mediates the acquired immune response, which plays an important role in host anti-microbial infection. TLR3 is a double-stranded RNA recognition receptor of virus. Activation can activate NF- 魏 B and IRF3 pathway to resist virus infection. In addition, activated TLR3 can also induce cell damage and damage the blood vessel barrier in vivo. The aim of this study was to interfere with the cell damage induced by TLR3 by analyzing the key signal molecule of TLR3 damage and trying to inhibit the key molecule. To provide a theoretical basis for clinical selection of related targets to delay the progression of infectious diseases. Methods RT-PCR was used to detect gene expression, flow cytometry was used to detect protein expression. Western blot was used to detect the activation of signal transduction molecules. 4PII / Annexin V staining was used to detect apoptosis level. Antibody neutralization and experiments were used to verify the correlation between signal molecules and apoptosis. The eukaryotic expression vector pcDNA3.1 / 螖 Np63 伪 was constructed and transfected into endothelial cells. The effect of 螖 Np63 伪 on apoptosis induced by TLR3 was detected. Results the HUVEC of human umbilical vein endothelial cells expressed high levels of TLR2O3O4 and 5 in human umbilical vein endothelial cells, low expression of TLR6 and 9, and no expression of TLR1 7MN 8 and 10. All TLR1-10 genes were expressed in human peripheral blood mononuclear cells as control. TLR3 ligand was used to express all TLR1-10 genes. Poly 鈪，

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