本文選題：TLR3　切入點：Poly　出處：《中南大學》2009年碩士論文　論文類型：學位論文

【摘要】： 目的:Toll樣受體(Toll-like receptors,TLRs)是病原相關分子模式識別受體,與相應配體結合后激活其下游信號轉導途徑,誘導炎癥反應和天然免疫應答,同時促進抗原提呈細胞的活化并介導獲得性免疫反應,從而在宿主抗微生物感染中發(fā)揮重要作用。TLR3是病毒雙鏈RNA識別受體,活化后能激活NF-κB和IRF3途徑來抵抗病毒感染。此外,TLR3活化后還能誘導細胞損傷,并破壞體內血管屏障引起病毒擴散,從而加速疾病的進程。本研究的目的是通過分析TLR3損傷的關鍵信號分子,嘗試抑制此關鍵分子來干預TLR3誘導的細胞損傷,為臨床選擇相關靶點延緩感染性疾病的進程提供理論基礎。 方法:1、RT-PCR檢測基因的表達。2、流式細胞術檢測蛋白質的表達。3、Western blot檢測信號轉導分子的活化。4、PI/Annexin V染色檢測細胞凋亡水平。5、抗體中和實驗驗證相關信號分子與細胞凋亡的相關性。6、構建真核表達載體pcDNA3.1+/ΔNp63α并轉染內皮細胞,檢測ΔNp63α對TLR3誘導細胞凋亡的影響。 結果:RT-PCR結果顯示人臍靜脈內皮細胞HUVEC較高表達Toll樣受體家族中的TLR2、3、4和5,低表達TLR6和9,不表達TLR1、7、8和10。作為對照人外周血單個核細胞表達所有TLR1-10基因。利用TLR3配體dsRNA的類似物PolyⅠ:C刺激HUVEC,結果顯示PolyⅠ:C上調TLR3基因表達并呈劑量依賴性,FACS分析結果顯示HUVEC細胞表達TLR3蛋白質。Western blot檢測發(fā)現PolyⅠ:C作用TLR3誘導了其下游信號分子NF-κB的活化,并劑量依賴性地上調細胞因子IL-1β、IL-6、IL-8、TNF-α、IFN-β的基因表達,表明HUVEC表達功能性的TLR3受體。此外,PolyⅠ:C處理的HUVEC細胞表現出與Staurosprine(一種凋亡誘導化學物質)處理后相似的形態(tài)學改變,PI/Annexin V染色顯示PolyⅠ:C誘導了劑量相關的細胞凋亡;這種作用由TLR3所介導,因為TLR3的中和抗體能顯著抑制PolyⅠ:C所誘導的細胞凋亡。PolyⅠ:C處理的HUVEC同時活化了caspase 8和9及其下游分子PARP,caspase和PARP抑制劑能顯著抑制PolyⅠ:C誘導的凋亡,表明TLR3通過內源性途徑(又稱線粒體途徑)和外源性途徑(又稱死亡受體途徑)誘導細胞凋亡。由于我們同期的研究還發(fā)現TLR3誘導的細胞凋亡受p53家族成員TAp63α的調控,因此我們構建了TAp63α的顯性負性異構體ΔNp63α的真核表達載體,轉染HUVEC后顯示其能顯著抑制PolyⅠ:C誘導的細胞凋亡。 結論:HUVEC表達功能性的TLR3,活化的TLR3通過內、外源性兩條途徑誘導HUVEC細胞凋亡。TAp63α的顯性負性突變異構體ΔNp63α降低PolyⅠ:C誘導的細胞凋亡,推測TAp63α在TLR3誘導的細胞凋亡中發(fā)揮重要作用。
[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 偽 was constructed and transfected into endothelial cells. The effect of 螖 Np63 偽 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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