Ras信号通路在HIV-1 Tat诱导ZO-1及脑啡肽酶破坏中的作用

发布时间：2018-03-03 09:37

本文选题：HIV-1　切入点：Tat　出处：《广西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:探讨Ras信号传导通路在人类免疫缺陷病毒-1型反式转录激活因子(HIV-1 transactivator of transcription,HIV-1 Tat)诱导血脑屏障(blood brain barrier,BBB)中紧密连接蛋白带状闭合蛋白-1(Zonula Occludens-1,ZO-1)及脑啡肽酶(neprilysin,NEP)破坏中的作用。方法:培养人脑微血管内皮细胞(human cerebral microvascular endothelium cells,HBEC-5i),以不同浓度HIV-1 Tat、Ras信号传导通路抑制剂法尼基硫代水杨酸(farnesylthiosalicylic acid,FTS)分别刺激细胞24h,并设立对照组,用四甲基偶氮唑盐(MTT法)检测其对细胞活力的影响。分别以HIV-1 Tat、FTS及FTS预处理细胞3h后再加入HIV-1 Tat共同作用24h后,以蛋白免疫印迹法(western blot,WB)及实时反转录聚合酶链式反应(real-time reverse transcription polymerase chain reaction,q RT-PCR)检测HIV-1 Tat诱导的紧密连接蛋白ZO-1及NEP(脑内降解β淀粉样蛋白amyloid-beta,Aβ的限速酶)的蛋白和mRNA表达的变化;分别以免疫荧光法(immunofluorescence,IF)检测HIV-1 Tat诱导的ZO-1及NEP免疫反应性的变化,以二氯二氢荧光素-乙酰乙酸酯(2′,7′-dichlorodihy-drofluorescein diacetate,DCFH-DA)荧光探针法检测细胞内活性氧(reactive oxygen species,ROS)的含量。同时,利用荧光标记的Aβ蛋白(Aβ(1-40)Hilyte)检测经过治疗后的细胞中外源性Aβ的沉积。结果:HIV-1 Tat、FTS浓度分别在1μg/mL和20μmol/L以下时对HBEC-5i活力无明显影响(P0.05);HIV-1 Tat诱导组的ZO-1及NEP蛋白(P0.01,P0.05)与mRNA(P0.01,P0.01)表达水平显著下调,蛋白免疫反应性降低,细胞内活性氧含量(P0.001)及Aβ蛋白沉积均增加。FTS预处理细胞3h可显著上调HIV-1 Tat诱导的ZO-1与NEP蛋白(P0.01,P0.05)及mRNA(P0.01,P0.01)的表达水平,增强ZO-1及NEP蛋白免疫反应性,降低细胞内活性氧含量及Aβ蛋白的沉积。结论:HIV-1 Tat可促进紧密连接蛋白ZO-1蛋白和mRNA下调而导致血脑屏障破坏,同时诱导脑内脑啡肽酶NEP蛋白和mRNA下调,促进细胞内活性氧的生成,可能导致Aβ在脑内沉积增加。阻断Ras信号传导通路可抑制HIV-1 Tat诱导的ZO-1和NEP破坏,减少ROS的产生,可能减少Aβ在脑内的沉积。
[Abstract]:Objective: to investigate the effect of Ras signal transduction pathway on the destruction of tight junction protein (-1Zonula Occludens-1Zonula Occludens-1ZO-1) and enkephalase neprilysin (NEP) in the blood brain barrier (BBB) induced by HIV-1 transactivator of transcriptional activator HIV-1 (HIV-1). Methods: human cerebral microvascular endothelium cells (HBEC-5iG) were cultured and stimulated with different concentrations of HIV-1 tyrosine Ras signaling pathway inhibitor, farnesylthiosalicylic acidic acid (FTSs), for 24 h, respectively, and the control group was set up. The cell viability was detected by tetramethylazolium tetrazolium. The cells were pretreated with HIV-1 tyrosine and FTS for 3 h and then treated with HIV-1 Tat for 24 h. Western blotDNA and real-time reverse transcription polymerase chain reactionQ RT-PCRs were used to detect the expression of HIV-1 Tat induced ZO-1 and NEP (amyloid-beta A 尾 rate-limiting enzyme in brain). Immunofluorescence assay was used to detect the changes of ZO-1 and NEP immunoreactivity induced by HIV-1 Tat, and the content of reactive oxygen speciesrose in cells was detected by fluorescence probe method of dichlorodihy-dichlorodihy-fluorescein diacetate (DCFH-DAA). The deposition of exogenous A 尾 in the treated cells was detected by fluorescence labeled A 尾 protein A 尾 1-40 (Hilyte). Results when the concentration of mol/L was below 1 渭 g / mL and 20 渭 mol/L, the expression of ZO-1 and NEP protein P0.01P0.05) and mRNA-P0.01P0.01P0.01in HIV-1 Tat induced group were not significantly affected by the concentration of FT-FTS, and the expression levels of NEP protein P0.01P0.05and mRNA-P0.01P0.01P0.01) were down-regulated in HIV-1 Tat induced group, respectively, when the concentration of FFTS was below 1 渭 g / mL and 20 渭 mol/L, respectively, there was no significant effect on the activity of HBEC-5i. The protein immunoreactivity decreased, the intracellular reactive oxygen content (P0.001) and A 尾 protein deposition increased. FTS-pretreated cells significantly up-regulated the expression levels of ZO-1 and NEP protein (P0.01P0.05) and mRNA-P0.01P0.01) induced by HIV-1 Tat for 3 h, and enhanced the immunoreactivity of ZO-1 and NEP protein. Conclusion HIV-1 Tat can promote the down-regulation of tight junction protein ZO-1 and mRNA and lead to the breakdown of blood-brain barrier, and induce the down-regulation of enkephalase NEP protein and mRNA. Blocking Ras signal transduction pathway can inhibit ZO-1 and NEP damage induced by HIV-1 Tat, decrease ROS production, and reduce A 尾 deposition in brain.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.91;R747.9

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