【摘要】：Ⅰ型人免疫缺陷病毒（HIV-1）編碼的反式激活蛋白（Tat）可通過(guò)多種途徑激活病毒的基因轉(zhuǎn)錄，組蛋白H3K27甲基化酶EZH2和去甲基化酶UTX在腫瘤形成、細(xì)胞分化和器官形成、病毒感染復(fù)制和潛伏感染等方面發(fā)揮重要作用。表沒(méi)食子兒茶素沒(méi)食子酸酯（EGCG）是綠茶中的主要茶多酚成分，具有抗氧化、抗炎、抗病毒的特性。本課題主要研究EZH2、UTX在Tat介導(dǎo)的HIV-1病毒長(zhǎng)末端重復(fù)序列（LTR）轉(zhuǎn)錄激活的作用及其潛在機(jī)制。同時(shí)探究了EGCG抑制Tat介導(dǎo)的LTR轉(zhuǎn)錄激活的作用及途徑。 應(yīng)用質(zhì)粒定點(diǎn)突變技術(shù)，構(gòu)建了一系列包括酶失活在內(nèi)的質(zhì)粒突變體；應(yīng)用多核活化半乳糖苷酶指示劑（MAGI）實(shí)驗(yàn)檢測(cè)轉(zhuǎn)染的Tat質(zhì)粒對(duì)TZM-bl細(xì)胞中LTR的激活程度。應(yīng)用實(shí)時(shí)定量PCR（RT-PCR）和western blot技術(shù)檢測(cè)mRNA和蛋白表達(dá)水平。 基因測(cè)序顯示質(zhì)粒突變體構(gòu)建成功；使用siRNA干擾EZH2的表達(dá)或EZH2的抑制劑DZNep增強(qiáng)了Tat蛋白介導(dǎo)的LTR的激活作用；細(xì)胞內(nèi)過(guò)表達(dá)EZH2質(zhì)粒及其突變體EZH2S21A、EZH2H694A則減弱了Tat蛋白這種作用。使用siRNA干擾UTX的表達(dá)減弱了Tat蛋白介導(dǎo)的LTR激活作用，，細(xì)胞內(nèi)過(guò)表達(dá)UTX質(zhì)粒增強(qiáng)了Tat蛋白的這種作用。Tat質(zhì)粒轉(zhuǎn)染不影響TZM-bl細(xì)胞中EZH2和UTX的mRNA水平，Tat質(zhì)粒轉(zhuǎn)染分別下調(diào)了TZM-bl細(xì)胞中EZH2的蛋白水平、H3K27me2和H3K27me3水平；Tat質(zhì)粒上調(diào)了UTX蛋白水平；Tat突變體Tat-C30G也有較弱的類似功能。同時(shí)發(fā)現(xiàn)EGCG能夠上調(diào)細(xì)胞核內(nèi)Nrf2蛋白水平，激活A(yù)MPK信號(hào)通路，抑制AKT信號(hào)通路，抑制了Tat介導(dǎo)的HIV-1LTR的轉(zhuǎn)錄激活。對(duì)EZH2、UTX、天然產(chǎn)物EGCG與Tat介導(dǎo)的轉(zhuǎn)錄激活關(guān)系的研究可以使我們對(duì)HIV-1的致病和潛伏感染機(jī)理有更深入的認(rèn)識(shí)，從而為HIV-1的治療方法和藥物篩選提供新的啟示。
[Abstract]:The transactivator protein (Tat) encoded by human immunodeficiency virus type I (HIV-1) can activate the gene transcription of the virus in a variety of ways. Histone H3K27 methylase EZH2 and demethylase UTX are involved in tumor formation, cell differentiation and organ formation. Viral infection replication and latent infection play an important role. Epigallocatechin gallate (EGCG) is the main tea polyphenol in green tea, which has the characteristics of antioxidation, anti-inflammation and anti-virus. In this study, we investigated the role of EZH2,UTX in the transcriptional activation of long terminal repeat (LTR) of HIV-1 virus mediated by Tat and its potential mechanism. At the same time, we explored the role and pathway of EGCG in inhibiting Tat mediated activation of LTR transcription. A series of plasmid mutants including enzyme inactivation were constructed by site-directed mutagenesis, and the activity of LTR in TZM-bl cells was detected by polynuclear activated galactosidase indicator (MAGI) assay. Real-time quantitative PCR (RT-PCR) and western blot techniques were used to detect the expression of mRNA and protein. Gene sequencing showed that the plasmid mutants were successfully constructed, that the expression of EZH2 was interfered by siRNA or that DZNep, the inhibitor of EZH2, enhanced the activation of LTR mediated by Tat protein, and that the overexpression of EZH2 plasmid and its mutant EZH2S21A,EZH2H694A weakened the effect of Tat protein. Using siRNA to interfere the expression of UTX attenuated the activation of LTR mediated by Tat protein, and the overexpression of UTX plasmid enhanced the effect of Tat protein. Tat plasmid transfection did not affect the mRNA levels of EZH2 and UTX in TZM-bl cells. The levels of EZH2 protein, H3K27me2 and H3K27me3 in TZM-bl cells were down-regulated by Tat plasmid transfection. Tat plasmid upregulated the level of UTX protein and Tat mutant Tat-C30G had weak similar function. At the same time, it was found that EGCG could up-regulate the level of Nrf2 protein in the nucleus, activate the AMPK signaling pathway, inhibit the AKT signal pathway, and inhibit the transcriptional activation of HIV-1LTR mediated by Tat. The study of the relationship between EZH2,UTX, natural product EGCG and transcriptional activation mediated by Tat can give us a deeper understanding of the pathogenesis and latent infection mechanism of HIV-1 and provide new inspiration for the treatment of HIV-1 and drug screening.
【學(xué)位授予單位】：北京工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R512.91

【參考文獻(xiàn)】

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1 滕競(jìng)飛;潘衛(wèi);;HIV-1 Tat蛋白的生物學(xué)特性及其致病效應(yīng)[J];中國(guó)生物制品學(xué)雜志;2009年04期

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