本文選題：TLR3　切入點(diǎn)：Poly　出處：《中南大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:Toll樣受體(Toll-like receptors,TLRs)是病原相關(guān)分子模式識(shí)別受體,與相應(yīng)配體結(jié)合后激活其下游信號(hào)轉(zhuǎn)導(dǎo)途徑,誘導(dǎo)炎癥反應(yīng)和天然免疫應(yīng)答,同時(shí)促進(jìn)抗原提呈細(xì)胞的活化并介導(dǎo)獲得性免疫反應(yīng),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(shí)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(dǎo)細(xì)胞損傷,并破壞體內(nèi)血管屏障引起病毒擴(kuò)散,從而加速疾病的進(jìn)程。本研究的目的是通過分析TLR3損傷的關(guān)鍵信號(hào)分子,嘗試抑制此關(guān)鍵分子來干預(yù)TLR3誘導(dǎo)的細(xì)胞損傷,為臨床選擇相關(guān)靶點(diǎn)延緩感染性疾病的進(jìn)程提供理論基礎(chǔ)。 方法:1、RT-PCR檢測(cè)基因的表達(dá)。2、流式細(xì)胞術(shù)檢測(cè)蛋白質(zhì)的表達(dá)。3、Western blot檢測(cè)信號(hào)轉(zhuǎn)導(dǎo)分子的活化。4、PI/Annexin V染色檢測(cè)細(xì)胞凋亡水平。5、抗體中和實(shí)驗(yàn)驗(yàn)證相關(guān)信號(hào)分子與細(xì)胞凋亡的相關(guān)性。6、構(gòu)建真核表達(dá)載體pcDNA3.1+/ΔNp63α并轉(zhuǎn)染內(nèi)皮細(xì)胞,檢測(cè)ΔNp63α對(duì)TLR3誘導(dǎo)細(xì)胞凋亡的影響。 結(jié)果:RT-PCR結(jié)果顯示人臍靜脈內(nèi)皮細(xì)胞HUVEC較高表達(dá)Toll樣受體家族中的TLR2、3、4和5,低表達(dá)TLR6和9,不表達(dá)TLR1、7、8和10。作為對(duì)照人外周血單個(gè)核細(xì)胞表達(dá)所有TLR1-10基因。利用TLR3配體dsRNA的類似物PolyⅠ:C刺激HUVEC,結(jié)果顯示PolyⅠ:C上調(diào)TLR3基因表達(dá)并呈劑量依賴性,FACS分析結(jié)果顯示HUVEC細(xì)胞表達(dá)TLR3蛋白質(zhì)。Western blot檢測(cè)發(fā)現(xiàn)PolyⅠ:C作用TLR3誘導(dǎo)了其下游信號(hào)分子NF-κB的活化,并劑量依賴性地上調(diào)細(xì)胞因子IL-1β、IL-6、IL-8、TNF-α、IFN-β的基因表達(dá),表明HUVEC表達(dá)功能性的TLR3受體。此外,PolyⅠ:C處理的HUVEC細(xì)胞表現(xiàn)出與Staurosprine(一種凋亡誘導(dǎo)化學(xué)物質(zhì))處理后相似的形態(tài)學(xué)改變,PI/Annexin V染色顯示PolyⅠ:C誘導(dǎo)了劑量相關(guān)的細(xì)胞凋亡;這種作用由TLR3所介導(dǎo),因?yàn)門LR3的中和抗體能顯著抑制PolyⅠ:C所誘導(dǎo)的細(xì)胞凋亡。PolyⅠ:C處理的HUVEC同時(shí)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(dǎo)的凋亡,表明TLR3通過內(nèi)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(dǎo)細(xì)胞凋亡。由于我們同期的研究還發(fā)現(xiàn)TLR3誘導(dǎo)的細(xì)胞凋亡受p53家族成員TAp63α的調(diào)控,因此我們構(gòu)建了TAp63α的顯性負(fù)性異構(gòu)體ΔNp63α的真核表達(dá)載體,轉(zhuǎn)染HUVEC后顯示其能顯著抑制PolyⅠ:C誘導(dǎo)的細(xì)胞凋亡。 結(jié)論:HUVEC表達(dá)功能性的TLR3,活化的TLR3通過內(nèi)、外源性兩條途徑誘導(dǎo)HUVEC細(xì)胞凋亡。TAp63α的顯性負(fù)性突變異構(gòu)體ΔNp63α降低PolyⅠ:C誘導(dǎo)的細(xì)胞凋亡,推測(cè)TAp63α在TLR3誘導(dǎo)的細(xì)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，

本文編號(hào)：1631456