TLR3上调TAP63α诱导细胞凋亡的分子机制

发布时间：2018-08-10 17:14

【摘要】： 目的:Toll样受体(Toll-like receptors,TLRs)是一类病原分子识别受体,其生物学功能主要是识别微生物感染,促进炎症细胞因子的合成与释放,介导炎症反应和先天性免疫;TLRs活化后还可以促进免疫细胞表达免疫分子,促进抗原提呈细胞的成熟,从而诱导机体的获得性免疫反应,因而TLRs又是机体介导天然免疫转向获得性免疫的桥梁。TLR3是机体识别病毒双链RNA(dsRNA)的受体,在体内抗病毒感染中发挥重要作用。一方面dsRNA作用于TLR3诱导免疫细胞的活化,促使免疫细胞产生抗病毒细胞因子如IFNα、β等,发挥抗病毒天然免疫作用;另一方面活化的TLR3可介导组织细胞(如血管内皮细胞)的损伤,从而破坏宿主的血管屏障如血脑屏障,促进病毒的扩散。本研究的目的是在前期研究的基础上进一步分析活化TLR3诱导人静脉内皮细胞(HUVEC)凋亡的分子机制,寻找TLR3诱导细胞凋亡信号转导途径中的关键调控分子,并尝试以此为靶点对TLR3诱导的细胞凋亡进行干预,为临床选择新的靶点控制病毒性疾病的发展提供理论依据。方法:(1)RT-PCR检测基因的表达;(2)ELISA检测分泌蛋白的水平;(3)PI-Annexin V染色流式细胞术检测细胞凋亡水平;(4)抗体中和实验验证细胞因子与细胞凋亡的相关性;(5)Western-Blotting检测细胞信号转导分子的表达和活化情况;(6)SiRNA干扰技术下调目的基因的表达。结果:ELISA结果表明dsRNA的类似物Poly(I:C)作用于HUVEC细胞,上调TNF-α的分泌,且呈剂量依赖性;重组的TNF-α诱导了HUVEC细胞中NF-κB的活化,但不能诱导HUVEC细胞的凋亡,而且TNF-α的中和抗体不能下调Poly(I:C)诱导的细胞凋亡,说明Poly(I:C)活化TLR3诱导的细胞凋亡与TNF-α无关。Poly(I:C)作用于HUVEC细胞后,RT-PCR检测发现TRAIL、DR4和DR5的表达上调,且呈剂量依赖性;PI-Annexin V染色证明TRAIL中和抗体部分下调了Poly(I:C)诱导的细胞凋亡,表明TRAIL-DR4/5启动外源性凋亡途径参与了TLR3诱导的细胞凋亡。进一步的研究表明Poly(I:C)下调Bcl-2家族中促生存蛋白Bcl-2的表达,上调Bcl-2家族中促凋亡蛋白Noxa和Puma的表达,同时上调细胞周期调控分子P21的表达,由于这些分子同时受肿瘤抑制基因P53或其家族成员的调控,提示P53或其家族成员参与了TLR3诱导的通过内源性途径介导的细胞损伤。然而RT-PCR结果显示活化TLR3并不引起P53的表达改变,P53的抑制剂也不影响Poly(I:C)诱导的细胞凋亡,表明P53没有参与Poly(I:C)诱导的损伤;当检测P53家族成员P63的表达时发现,Poly(I:C)上调TAP63α的表达;而且SiRNA抑制TAP63α蛋白的表达后,抑制了Bcl-2的下调,抑制了Noxa的上调,抑制了Caspase8、Caspase 9和PARP的活化,并显著抑制Poly(I:C)诱导的细胞凋亡,表明TAP63α是调控TLR3诱导细胞损伤的关键分子。结论:TNF-α不参与TLR3诱导的HUVEC细胞凋亡的外源性途径的启动;TRAIL-DR4/DR5作用机制参与了TLR3诱导的HUVEC细胞凋亡的细胞外途径的启动;TLR3通过下调Bcl-2家族中的促生存分子Bcl-2表达,上调促凋亡分子Noxa表达上调启动内源性细胞凋亡。P53家族成员TAp63α是TLR3诱导的HUVEC细胞凋亡的关键调控分子。
[Abstract]:AIM: Toll-like receptors (TLRs) are a class of pathogenic molecular recognition receptors. Their biological functions are mainly to recognize microbial infections, promote the synthesis and release of inflammatory cytokines, mediate inflammation and innate immunity. Activation of TLRs can also promote the expression of immune molecules in immune cells and promote the formation of antigen presenting cells. TLRs are the bridge between the innate immunity and the acquired immunity. TLR3 is the receptor that recognizes the double-stranded RNA (dsRNA) of the virus and plays an important role in anti-virus infection. On the one hand, dsRNA acts on TLR3 to induce the activation of immune cells and induce immune cells to produce anti-virus. Viral cytokines such as IFN alpha and beta play a natural immune role against viruses. On the other hand, activated TLR3 can mediate the damage of tissue cells (such as vascular endothelial cells), thereby destroying the host vascular barrier such as blood-brain barrier, and promoting the spread of viruses. The purpose of this study is to further analyze the role of activated TLR3 in inducing human beings on the basis of previous studies. The molecular mechanism of apoptosis in vein endothelial cells (HUVEC) is to find the key regulators in the signal transduction pathway of TLR3-induced apoptosis, and try to interfere with the apoptosis induced by TLR3 as a target, so as to provide theoretical basis for clinical selection of new targets to control the development of viral diseases.
Methods: (1) RT-PCR detection of gene expression; (2) ELISA detection of secretory protein; (3) PI-Annexin V staining flow cytometry detection of apoptosis; (4) antibody neutralization test to verify the correlation between cytokines and apoptosis; (5) Western-Blotting detection of cell signal transduction molecule expression and activation; (6) SiRNA interference technology under the SiRNA interference. The expression of target genes.
Results: ELISA showed that poly (I:C), a dsRNA analogue, increased the secretion of TNF-alpha in HUVEC cells in a dose-dependent manner; recombinant TNF-alpha induced the activation of NF-kappa B in HUVEC cells, but could not induce the apoptosis of HUVEC cells, and the neutralizing antibody against TNF-alpha could not down-regulate the apoptosis induced by poly (I:C), indicating that poly (I:C) activated TLR. The expression of TRAIL, DR4 and DR 5 was up-regulated in a dose-dependent manner after poly (I:C) acted on HUVEC cells. PI-Annexin V staining showed that TRAIL neutralizing antibody partially down-regulated the apoptosis induced by poly (I:C), suggesting that TRAIL-DR4/5 initiated exogenous apoptosis pathway participated in TLR3-induced fine cells. Further studies have shown that Poly(I:C) down-regulates the expression of Bcl-2, up-regulates the expression of Noxa and Puma, and up-regulates the expression of P21, which are both regulated by tumor suppressor gene P53 or its family members, suggesting the formation of P53 or its family. However, the results of RT-PCR showed that activated TLR3 did not change the expression of P53, and the inhibitor of P53 did not affect the apoptosis induced by Poly (I:C), suggesting that P53 did not participate in the injury induced by Poly (I:C). Poly (I:C) up-regulated the expression of P63 in P53 family members. Moreover, SiRNA inhibited the down-regulation of Bcl-2, inhibited the up-regulation of Noxa, inhibited the activation of Caspase 8, Caspase 9 and PARP, and significantly inhibited the apoptosis induced by Poly (I:C), suggesting that TAP63a was a key molecule in regulating TLR3-induced cell injury.
Conclusion: TNF-alpha does not participate in the initiation of exogenous pathway of TLR3-induced apoptosis of HUVEC cells; the mechanism of TRAIL-DR4/DR5 participates in the initiation of extracellular pathway of TLR3-induced apoptosis of HUVEC cells; TLR3 up-regulates the expression of Noxa, a pro-apoptotic molecule, by down-regulating the expression of Bcl-2 in the Bcl-2 family. 3 family member TAp63 alpha is a key regulator of TLR3 induced apoptosis in HUVEC cells.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

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