【摘要】：研究背景： 細(xì)胞骨架踝蛋白(talin1)參與HIV-1入侵和突觸形成過程,并且能夠抑制HIV-1感染,但具體機(jī)制不明。我們通過差異蛋白質(zhì)組學(xué)的方法,發(fā)現(xiàn)talin1在未經(jīng)HARRT治療的HIV-1感染者PBMCs中降解成一個片段,經(jīng)過質(zhì)譜鑒定該片段位于talin1C末端,約38kDa,含有整合素(intergrin)及actin結(jié)合位點,且進(jìn)一步研究發(fā)現(xiàn)該片段與病毒載量負(fù)相關(guān)。通過生物信息學(xué)手段對鑒定的差異蛋白進(jìn)行相互作用分析,結(jié)果顯示,talin1參與了HIV-1功能基因pol和vif調(diào)控的網(wǎng)絡(luò),可見talin1片段在HIV-1感染過程中發(fā)揮重要作用,研究talin1在HIV-1感染中發(fā)生片段化及其抑制HIV-1感染的機(jī)制,有助于尋找介導(dǎo)HIV-1一宿主細(xì)胞相互作用的關(guān)鍵分子,為抗HIV-1藥物研究提供藥物新靶點。此外,HIV-1病毒的潛伏可能與HIV-1的入侵,整合以及復(fù)制逆轉(zhuǎn)錄等多個過程相關(guān),潛伏機(jī)制非常復(fù)雜。而在HIV-1的入侵,整合及逆轉(zhuǎn)錄等過程,細(xì)胞質(zhì)膜蛋白質(zhì)都發(fā)揮重要作用,研究HIV-1潛伏過程中細(xì)胞質(zhì)膜蛋白質(zhì)的表達(dá)變化能為發(fā)現(xiàn)HIV-1潛伏機(jī)制及發(fā)現(xiàn)HIV-1潛伏標(biāo)志物提供一定的線索。 研究目的： 研究talin1片段化與HIV-1感染的相關(guān)性,探討talin1影響HIV感染分子機(jī)制,篩選HIV潛伏細(xì)胞模型中的質(zhì)膜標(biāo)志蛋白質(zhì),為了解HIV-宿主細(xì)胞相互作用的分子機(jī)制提供幫助。 方法： 1、構(gòu)建HIV假病毒感染Tzm-b1細(xì)胞模型,研究talin1片斷化與HIV感染的時間依賴關(guān)系；構(gòu)建凋亡細(xì)胞模型,檢驗talin1片段化與細(xì)胞凋亡的相關(guān)性； 2、不同疾病患者PBMCs樣本中檢驗talin1片段化的表達(dá)； 3、通過電擊導(dǎo)入talin1蛋白片段及過表達(dá)38kDa talin1片段研究該38kDa talin1對HIV-1感染的影響； 4、通過iRNA干擾構(gòu)建talin1缺失株,研究talin1缺失對HIV-1感染的影響,并通過蛋白質(zhì)組學(xué)的方法探討可能參與talin1片段化過程的相互作用蛋白質(zhì)。 5、提取HIV-1潛伏細(xì)胞模型及其對照細(xì)胞的細(xì)胞質(zhì)膜蛋白質(zhì),通過二維凝膠電泳分離蛋白,使用ImageMaster2D Platinum6.0進(jìn)行圖像分析軟件分析差異蛋白質(zhì)點。 結(jié)果： 1、Talin1在HIV-1感染初期就發(fā)生片段化,并且talin1片段化與細(xì)胞凋亡沒有直接相關(guān)性； 2、Talin1在HBV患者中也發(fā)生了片段化,并在體外MLV病毒感染細(xì)胞模型中也檢測到talin1片段,且從感染初期就發(fā)生片段化； 3、點擊導(dǎo)入talin1蛋白片段能夠明顯抑制HIV-1的感染；過表達(dá)38kDatalin1片段也能抑制HIV-1的感染； 4、Talin1缺失能夠促進(jìn)HIV-1感染,talin1頭部50kDa的功能與C端38kDa的功能相反,它能促進(jìn)HIV-1感染； 5、HIV-1潛伏細(xì)胞模型質(zhì)膜蛋白質(zhì)組學(xué)研究發(fā)現(xiàn)16個差異蛋白質(zhì)點,通過鑒定后得到12個非冗余蛋白質(zhì),其中3個蛋白質(zhì)在HIV-1潛伏細(xì)胞中表達(dá)下調(diào),9個蛋白質(zhì)在HIV-1潛伏細(xì)胞中不表達(dá)； 結(jié)論： 初步研究talin1片斷化與HIV感染的關(guān)系：HIV感染導(dǎo)致talin1片斷化,talin1片斷具有抗HIV的活性；Talin1片斷以及在HIV潛伏感染細(xì)胞模型中發(fā)現(xiàn)的APR3可能為潛在的抗病毒藥物或者藥物靶標(biāo)。
[Abstract]:Study Background: The cytoskeleton ankle (talin1) is involved in the process of HIV-1 invasion and synapse formation and is capable of inhibiting HIV-1 infection, but specific mechanisms We found that talin1 was degraded into a fragment from PBMCs of the HIV-1 infected person without the HARRT, and the fragment was identified by mass spectrometry at the end of the tinin 1C, about 38kDa, containing the integrin and actin binding. site, and further study found that the fragment was negative with viral load The results showed that talin1 was involved in the control of the HIV-1 function gene pol and vif, and it was found that the talin1 fragment played an important role in the process of HIV-1 infection. The role of talin1 in the pathogenesis of HIV-1 infection and its inhibition of HIV-1 infection can help to find the key molecules to mediate the interaction of HIV-1 host cells and provide new drugs for anti-HIV-1 drug research. In addition, the latent mechanism of HIV-1 virus can be related to multiple processes of HIV-1 invasion, integration and reverse transcription, and the latent mechanism is very important. In the process of HIV-1 invasion, integration and reverse transcription, the cytoplasmic membrane protein plays an important role in the study of the expression of the cytoplasmic membrane protein in the HIV-1 latent process, which can be used to find the latent mechanism of HIV-1 and to find the latent mark of HIV-1. A clue. Objective: To study the relationship between talin1 fragmentation and HIV-1 infection, to study the molecular mechanism of talin1 on HIV infection, to screen the membrane marker proteins in the HIV latent cell model and to understand the interaction of HIV-host cells. molecular machine Methods: 1. The model of Tzm-b1 cells infected with HIV pseudovirus was constructed to study the time-dependent relationship between talin1 and HIV infection. the correlation between the fragmentation of n1 and the apoptosis of cells; 2. PBMCs of patients with different diseases sample the expression of the talin1 fragment was examined in. 3, the 38 kDa talin1 fragment was introduced by electric shock and the 38 kDa talin1 fragment was overexpressed the effect of kDa talin1 on HIV-1 infection; 4. Construction of the talin1 deletion via iRNA interference The effect of the deletion of talin1 on HIV-1 infection and the method of proteomics It is possible to take part in the interaction protein of the tinin1 fragmentation process. 5. Extract the cytoplasmic membrane proteins of the HIV-1 latent cell model and its control cells, isolate the proteins by two-dimensional gel electrophoresis, and use the ImageMaster2D P lat Image analysis software was used to analyze the difference protein points in inum6.0. Results: 1, Talin1 was infected with HIV-1. there was a fragmentation in the early stage, and the talin1 fragmentation was not directly related to cell apoptosis; 2, the Talin1 was also in the HBV patient Fragmentation occurs and in vitro ML the talin1 fragment was also detected in the V-infected cell model, and fragmentation occurred in the early stage of infection. the infection of HIV-1 is made; the overexpression of the 38kdatalin1 fragment can also inhibit the infection of HIV-1; 4, the absence of the Talin1 can promote the HIV-1 infection, The function of the talin1 head 50kDa was opposite to that of the C-terminal 38kDa, which can promote the HIV-1 infection; 5, the membrane proteome of the HIV-1 latent cell model was found to be 16 different protein points, and 12 non-redundant proteins were obtained by the identification. the mass, which The expression of 3 proteins in HIV-1 latent cells was down-regulated and 9 of the proteins were not expressed in HIV-1 latent cells. Conclusion: The relationship between talin1 fragmentation and HIV infection is studied in this paper.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R512.91

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