本文關(guān)鍵詞： RBBP4 HIV LTR HDAC NR2F1　出處：《浙江大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：背景由人類免疫缺陷病毒(human immunodeficiency virus, HIV)所導(dǎo)致的艾滋病是目前最具有威脅性的傳染病之一。雖然高效的抗逆轉(zhuǎn)錄病毒治療(highly active anti-retroviral therapy, HAART)能有效的抑制HIV病毒的復(fù)制并使病毒載量降低至檢測下限,但是由于HIV潛伏庫的存在,導(dǎo)致HIV不能被徹底清除,因此當(dāng)前HIV潛伏庫被認(rèn)為是從體內(nèi)徹底清除HIV的最后壁壘。HIV潛伏狀態(tài)本質(zhì)上是一種可逆的轉(zhuǎn)錄沉默狀態(tài),這種狀態(tài)的產(chǎn)生和維持涉及多種宿主轉(zhuǎn)錄抑制因子及表觀修飾復(fù)合體。結(jié)合在HIV前病毒基因組長末端重復(fù)序列5'LTR (long terminal repeat)上的宿主轉(zhuǎn)錄抑制因子及其募集的組蛋白脫乙�；窰DACs (histone deacetylase)等表觀修飾復(fù)合體在HIV的轉(zhuǎn)錄沉默和潛伏的建立維持中起著重要作用。視網(wǎng)膜母細(xì)胞瘤結(jié)合蛋白RBBP4 (retinoblastoma binding protein 4),作為組蛋白分子伴侶,通過與HDACs等表觀修飾復(fù)合體協(xié)同作用,可參與基因的轉(zhuǎn)錄沉默。但作為與HDACs密切關(guān)聯(lián)的RBBP4分子,它在HIV的轉(zhuǎn)錄以及潛伏中的作用還未見報道。因此,本文將對RBBP4在HIV轉(zhuǎn)錄和潛伏中的作用及機(jī)制進(jìn)行研究。目的1.研究RBBP4在正常對照者、臨床HIV感染者和治療者外周血單個核細(xì)胞PBMC (peripheral blood mononuclear cells)中的表達(dá)變化,并在實(shí)驗(yàn)室HIV感染的CEM-ss和TZM-bl細(xì)胞模型上對RBBP4的表達(dá)變化進(jìn)行驗(yàn)證；2.研究RBBP4表達(dá)的改變對HIV產(chǎn)生的作用,并在HIV的轉(zhuǎn)錄環(huán)節(jié)研究RBBP4對HIV LTR介導(dǎo)的轉(zhuǎn)錄調(diào)控以及RBBP4與HIV LTR啟動子的結(jié)合；3.進(jìn)一步在HIV的感染和潛伏模型中研究RBBP4對HIV轉(zhuǎn)錄和潛伏作用的機(jī)制,為HIV潛伏庫的清除提供實(shí)驗(yàn)數(shù)據(jù)及科學(xué)依據(jù)。方法1.首先利用熒光定量PCR檢測RBBP4分子在臨床HIV感染者(30例)、HIV治療者(30例)和健康對照(30例)的PBMCs中的表達(dá)變化。并在實(shí)驗(yàn)室HIV感染的CEM-ss和TZM-bl細(xì)胞模型上,利用熒光定量PCR、Western blot和免疫熒光技術(shù),對RBBP4的表達(dá)變化進(jìn)行定性和定量檢測。2.通過雙熒光素酶報告系統(tǒng)檢測HIV LTR啟動轉(zhuǎn)錄的螢火蟲熒光素酶活性,來研究RBBP4對HIV LTR啟動子的轉(zhuǎn)錄調(diào)控；利用凝膠遷移實(shí)驗(yàn)(electrophoretic mobility shift assay, EMSA研究RBBP4與HIV LTR上NF-κB(-115～-75)和NR2F1(-345--319)結(jié)合位點(diǎn)的結(jié)合。3.在293T細(xì)胞與pNL4-3轉(zhuǎn)染的細(xì)胞模型中,利用染色質(zhì)免疫共沉淀(chrmatin immunoprecipitation analysis, ChIP)研究RBBP4對HDAC1/2和NR2F1在LTR上結(jié)合的影響,并在HIV潛伏和激活的CEM-Bru細(xì)胞中研究RBBP4、 HDAC1、HDAC2和NR2F1在LTR上的結(jié)合變化。結(jié)果1.發(fā)現(xiàn)RBBP4在臨床HIV感染者中的表達(dá)明顯高于HIV治療成功者和正常對照(P0.01；P0.01)；在實(shí)驗(yàn)室HIV感染的CEM-ss和TZM-bl細(xì)胞模型中,RBBP4 mRNA和蛋白的表達(dá)均明顯高于未感染的對照細(xì)胞,其結(jié)果與臨床發(fā)現(xiàn)一致；并進(jìn)一步發(fā)現(xiàn)在CEM-ss和TZM-bl細(xì)胞模型中將RBBP4干擾表達(dá)后再感染HIV,發(fā)現(xiàn)HIV病毒顆粒的產(chǎn)生明顯增加；2.過表達(dá)RBBP4可減少上清HIV病毒顆粒、病毒載量、單間接轉(zhuǎn)錄本、多剪接轉(zhuǎn)錄本和未剪接轉(zhuǎn)錄本的產(chǎn)生；進(jìn)一步的研究發(fā)現(xiàn)RBBP4可抑制HIV LTR啟動的本底轉(zhuǎn)錄以及NF-κB和Tat誘導(dǎo)的轉(zhuǎn)錄；RBBP4可分別與HIV LTR(-454～+181)上的NF-κB結(jié)合位點(diǎn)(-115～-75)和NR2F1結(jié)合位點(diǎn)(-345～-319)結(jié)合。3. RBBP4在HIV LTR上結(jié)合的變化能影響NR2F1、HDAC1/2在LTR上結(jié)合,即RBBP4在HIV LTR上的結(jié)合增加,NR2F1、HDAC1/2在LTR上的結(jié)合也增加,組蛋白乙酰化水平降低,最后導(dǎo)致HIV上清P24和病毒顆粒的產(chǎn)生減少；在HIV潛伏的CEM-Bru細(xì)胞中,RBBP4、NR2F1、HDAC1/2在HIV LTR的結(jié)合明顯高于在激活狀態(tài)下的結(jié)合；在潛伏細(xì)胞中將RBBP4干擾表達(dá)后能促使HIV起始轉(zhuǎn)錄本增加。結(jié)論1.首次發(fā)現(xiàn)RBBP4在未經(jīng)治療的HIV感染者中表達(dá)升高,并在HIV感染的TZM-bl和CEM-ss細(xì)胞中發(fā)現(xiàn)RBBP4的表達(dá)升高,表明HIV感染可誘導(dǎo)RBBP4表達(dá)增加；2. RBBP4可抑制HIV LTR介導(dǎo)的本底轉(zhuǎn)錄；RBBP4可抑制由NF-κB和Tat誘導(dǎo)激活的HIV LTR轉(zhuǎn)錄；并能與LTR上的NF-κB結(jié)合位點(diǎn)(-115～-75)和NR2F1結(jié)合位點(diǎn)(-345～-319)結(jié)合；3. RBBP4通過影響轉(zhuǎn)錄因子NR2F1和組蛋白脫乙酰酶HDAC1/2在啟動子上的結(jié)合來抑制HIV的轉(zhuǎn)錄和病毒顆粒的產(chǎn)生,并與NR2F1和HDAC1/2共同維持HIV的潛伏狀態(tài)。
[Abstract]:The background by the human immunodeficiency virus (human immunodeficiency, virus, HIV) caused by AIDS is one of the most threatening infectious disease. Although antiretroviral treatment (highly active anti-retroviral therapy, HAART) can effectively inhibit the replication of the HIV virus and the virus load to reduce the detection limit, but because of latent HIV the library exists, resulting in HIV cannot be completely eliminated, so the latent HIV library is considered to be.HIV the last barrier completely cleared from the body of HIV latency is essentially a reversible transcription silence state, this state and maintenance involves a variety of host transcriptional inhibitor and epigenetic modification combined with in front of the HIV complex. Virus long terminal repeat 5'LTR (long terminal repeat) on the inhibition of host transcription factor and recruitment of histone deacetylase HDACs (histo NE deacetylase) and other epigenetic transcriptional silencing complex in HIV and latent building plays an important role in maintaining. Retinoblastoma binding protein RBBP4 (retinoblastoma binding protein 4), as a histone chaperone, through epigenetic modification and HDACs complex synergistic effect, can participate in the transcriptional silencing of genes. But as RBBP4 molecules are closely associated with the HDACs, it has not been reported in HIV transcription and the latent role. Therefore, this paper studies RBBP4 in HIV transcription and latent in effect and mechanism. To study on 1. RBBP4 in normal controls, PBMC peripheral blood mononuclear cells and the clinical treatment of HIV infection outside (peripheral blood mononuclear cells) expression, and in the Laboratory of CEM-ss HIV infection and TZM-bl cell model on the expression of RBBP4 to test the expression of 2. RBBP4; the change of research The effects of HIV, HIV and LTR in RBBP4 to the study of transcription of HIV transcription regulation and RBBP4 with HIV LTR promoter binding; a further 3. in HIV infection and a potential mechanism model of RBBP4 on HIV transcription and latent function, provide experimental data and scientific basis for the removal of the latent reservoir HIV methods 1.. Using fluorescence quantitative PCR detection of RBBP4 molecules in clinical HIV infection (30 cases), HIV treatment (30 cases) and healthy controls (n = 30). The expression of PBMCs and CEM-ss in the laboratory of HIV infection and TZM-bl cell model, using fluorescence quantitative PCR, Western and blot immunofluorescence technique. The expression changes of RBBP4 were qualitative and quantitative detection of.2. by dual luciferase reporter assay HIV LTR promoter firefly luciferase activity to transcription, transcription of RBBP4 promoter of HIV LTR; the use of gel migration Electrophoretic mobility shift assay (shift experiment, EMSA RBBP4 and HIV LTR NF- of kappa B (-115 ~ -75) and NR2F1 (-345--319) combined with.3. in the cell model of 293T cells transfected with pNL4-3 loci, using chromatin immunoprecipitation (chrmatin immunoprecipitation analysis, ChIP) the influence of RBBP4 on HDAC1/2 and NR2F1 in the LTR with the HDAC1 and RBBP4 in the HIV study, latent and activated CEM-Bru cells, HDAC2 and NR2F1 in combination with the change of LTR. The results showed the expression of RBBP4 in 1. clinical HIV infection were significantly higher than in HIV treatment and normal control (P0.01; P0.01); in the laboratory of CEM-ss HIV infection the TZM-bl cell model, the expression of RBBP4 protein and mRNA were significantly higher than that of uninfected control cells, the result is consistent with clinical findings; and further found in CEM-ss and TZM-bl cell model, RBBP4 interference HIV expression after infection, HIV virus particles produced significantly increased 2.; overexpression of RBBP4 can reduce the supernatant of HIV virus particles, viral load, single connected transcripts, multiple splicing transcripts and spliced transcripts did not produce; further study found that RBBP4 can inhibit the HIV LTR promoter of the basal transcription and NF- K B and Tat induced transcription; RBBP4 and HIV LTR respectively (-454 ~ +181) of the NF- B binding site (-115 ~ -75) and NR2F1 binding sites (-345 ~ -319) combined with.3. RBBP4 based on HIV LTR can affect NR2F1, HDAC1/2 in LTR with RBBP4 in HIV LTR combined with NR2F1, HDAC1/2 increased in LTR with increased histone acetylation levels decreased, finally leads to the generation of HIV supernatant of P24 and virus particles decreased; in HIV latent CEM-Bru cells, RBBP4, NR2F1, HDAC1/2 in HIV combined with LTR was significantly higher than that in the activated state The combination of HIV can promote the initiation of transcription; increase in latency in cell RBBP4 interference expression. Conclusion 1. we found that the expression of RBBP4 in untreated HIV infection increased, and in HIV infected TZM-bl and CEM-ss cells showed increased expression of RBBP4, suggesting that HIV infection can induce increased expression of RBBP4; 2. RBBP4 can inhibit HIV mediated LTR RBBP4 can inhibit the basal transcription; HIV LTR transcription induced by NF- kappa B and Tat activation; and binding sites and LTR NF- kappa B (-115 ~ -75) and NR2F1 binding sites (-345 ~ -319) combined with 3. RBBP4; through the influence of transcription factor NR2F1 and histone deacetylase HDAC1/2 promoter to inhibit transcription and HIV virus production, and NR2F1 and HDAC1/2 to maintain HIV latency.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R512.91

【相似文獻(xiàn)】

相關(guān)期刊論文 前1條

1 夏夢;劉明兮;周濤;何輝;萬茹;霍然;;小鼠生發(fā)泡期卵母細(xì)胞成熟過程中轉(zhuǎn)錄沉默機(jī)制的探討[J];南京醫(yī)科大學(xué)學(xué)報(自然科學(xué)版);2011年07期

相關(guān)博士學(xué)位論文 前1條

1 王娟;RBBP4在HIV轉(zhuǎn)錄與潛伏中的作用及機(jī)制研究[D];浙江大學(xué);2016年

，

本文編號：1554623