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  本文選題：HIV-1　切入點：Tat　出處：《廣西醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:探討Ras信號傳導通路在人類免疫缺陷病毒-1型反式轉(zhuǎn)錄激活因子(HIV-1 transactivator of transcription,HIV-1 Tat)誘導血腦屏障(blood brain barrier,BBB)中緊密連接蛋白帶狀閉合蛋白-1(Zonula Occludens-1,ZO-1)及腦啡肽酶(neprilysin,NEP)破壞中的作用。方法:培養(yǎng)人腦微血管內(nèi)皮細胞(human cerebral microvascular endothelium cells,HBEC-5i),以不同濃度HIV-1 Tat、Ras信號傳導通路抑制劑法尼基硫代水楊酸(farnesylthiosalicylic acid,FTS)分別刺激細胞24h,并設立對照組,用四甲基偶氮唑鹽(MTT法)檢測其對細胞活力的影響。分別以HIV-1 Tat、FTS及FTS預處理細胞3h后再加入HIV-1 Tat共同作用24h后,以蛋白免疫印跡法(western blot,WB)及實時反轉(zhuǎn)錄聚合酶鏈式反應(real-time reverse transcription polymerase chain reaction,q RT-PCR)檢測HIV-1 Tat誘導的緊密連接蛋白ZO-1及NEP(腦內(nèi)降解β淀粉樣蛋白amyloid-beta,Aβ的限速酶)的蛋白和mRNA表達的變化;分別以免疫熒光法(immunofluorescence,IF)檢測HIV-1 Tat誘導的ZO-1及NEP免疫反應性的變化,以二氯二氫熒光素-乙酰乙酸酯(2′,7′-dichlorodihy-drofluorescein diacetate,DCFH-DA)熒光探針法檢測細胞內(nèi)活性氧(reactive oxygen species,ROS)的含量。同時,利用熒光標記的Aβ蛋白(Aβ(1-40)Hilyte)檢測經(jīng)過治療后的細胞中外源性Aβ的沉積。結(jié)果:HIV-1 Tat、FTS濃度分別在1μg/mL和20μmol/L以下時對HBEC-5i活力無明顯影響(P0.05);HIV-1 Tat誘導組的ZO-1及NEP蛋白(P0.01,P0.05)與mRNA(P0.01,P0.01)表達水平顯著下調(diào),蛋白免疫反應性降低,細胞內(nèi)活性氧含量(P0.001)及Aβ蛋白沉積均增加。FTS預處理細胞3h可顯著上調(diào)HIV-1 Tat誘導的ZO-1與NEP蛋白(P0.01,P0.05)及mRNA(P0.01,P0.01)的表達水平,增強ZO-1及NEP蛋白免疫反應性,降低細胞內(nèi)活性氧含量及Aβ蛋白的沉積。結(jié)論:HIV-1 Tat可促進緊密連接蛋白ZO-1蛋白和mRNA下調(diào)而導致血腦屏障破壞,同時誘導腦內(nèi)腦啡肽酶NEP蛋白和mRNA下調(diào),促進細胞內(nèi)活性氧的生成,可能導致Aβ在腦內(nèi)沉積增加。阻斷Ras信號傳導通路可抑制HIV-1 Tat誘導的ZO-1和NEP破壞,減少ROS的產(chǎn)生,可能減少Aβ在腦內(nèi)的沉積。
[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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R512.91;R747.9

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