本文選題：Ⅰ型單純皰疹病毒(HSV-1) + HTRP��； 參考：《中國協(xié)和醫(yī)科大學(xué)》2010年碩士論文

【摘要】： Ⅰ型單純皰疹病毒(HSV-1)是具有較強傳染性,嚴(yán)重危害人類健康的病原體,人類是其唯一的宿主,至今尚無相應(yīng)疫苗問世。作為一種結(jié)構(gòu)復(fù)雜,而且在基因轉(zhuǎn)錄復(fù)制過程中表現(xiàn)明確級聯(lián)效應(yīng)的雙鏈DNA病毒,HSV-1在與細(xì)胞的相互作用過程中呈現(xiàn)出了不同層次的特點。對于HSV-1病毒結(jié)合受體所導(dǎo)致的細(xì)胞早期基因產(chǎn)物功能的分析,不僅能加深對HSV-1和細(xì)胞之間相互作用的理解,也有助于闡明細(xì)胞中未知功能蛋白的作用方式及其分子機理。在此領(lǐng)域中,我們的前期研究發(fā)現(xiàn)HSV-1與人成纖維細(xì)胞KMB-17株表面受體結(jié)合后,能夠刺激細(xì)胞基因表達(dá)水平發(fā)生差異性的改變。同時,我們利用mRNA差異顯示和蛋白2D差異技術(shù)所特異誘導(dǎo)的細(xì)胞相關(guān)分子做了較為深入的研究,并從中篩選克隆到了一些新基因。本研究的htrp基因,即為上述研究工作中所克隆的功能未知的基因之一(基因登錄號：AF442486)。htrp所編碼蛋白HTRP(human transcription regulator protein)作為一個與細(xì)胞轉(zhuǎn)錄調(diào)控相關(guān)的蛋白分子,表現(xiàn)出能夠與轉(zhuǎn)錄輔助抑制復(fù)合物mSin3A的成分之一SAP30 (mSin3 A Association Protein, SAP30)之間相互作用,并由此促進(jìn)HSV-1感染細(xì)胞死亡的生物學(xué)效應(yīng)。 本課題圍繞著HTRP分子與SAP30分子在細(xì)胞與宿主之間的相互作用中的生物學(xué)功能及分子機制展開研究,揭示了HTRP對HSV-1病毒的普遍轉(zhuǎn)錄抑制效應(yīng),進(jìn)一步證實了HTRP與SAP30的轉(zhuǎn)錄抑制協(xié)同效應(yīng),并深入探討了兩者相互作用與轉(zhuǎn)錄抑制的密切聯(lián)系及其分子機制。首先我們利用上調(diào)表達(dá)HTRP的細(xì)胞環(huán)境來研究其對病毒復(fù)制增殖的影響,結(jié)果顯示HTRP可以抑制感染不同時期的病毒TK、gC基因的轉(zhuǎn)錄活性,且隨著時間的延長而抑制越明顯。接著,在HTRP和SAP30同時表達(dá)上調(diào)的細(xì)胞環(huán)境下,進(jìn)一步證實了兩者對TK、gC基因的轉(zhuǎn)錄抑制表現(xiàn)出協(xié)同效應(yīng)。有關(guān)細(xì)胞分子生物學(xué)的研究資料已經(jīng)證明,SAP30作為細(xì)胞內(nèi)轉(zhuǎn)錄調(diào)控的輔抑制復(fù)合物中的重要結(jié)構(gòu)成分之一,具有介導(dǎo)組蛋白去乙�；�(hi stone deacetyltransferases, HDAC)結(jié)合至該復(fù)合物的功能,我們的實驗表明HTRP和SAP30則可以通過使染色質(zhì)組蛋白的去乙�；緩綄崿F(xiàn)對酶基因的轉(zhuǎn)錄抑制。為了進(jìn)一步驗證HTRP與SAP30對病毒基因的協(xié)同抑制作用的分子機制,在雙熒光素酶檢測系統(tǒng)中加入HDACs抑制劑TSA的實驗證明,這種協(xié)同作用是通過HDAC酶活性的改變而實現(xiàn)的。同時,使用染色質(zhì)免疫沉淀(Chromatin Immunoprecipitation, ChIP)實驗表明,由于HTRP與SAP30的結(jié)合而導(dǎo)致HDACs活性的改變,使得HDACs酶增強了組蛋白H3分子第14和9位賴氨酸的去乙酰化水平。 基于上述實驗結(jié)果,我們可以針對HSV-1感染人成纖維細(xì)胞所特異上調(diào)的細(xì)胞蛋白分子HTRP的具體分子生物學(xué)作用機理提出如下模型：HTRP作為一種細(xì)胞在HSV-1感染時反應(yīng)性上調(diào)的轉(zhuǎn)錄調(diào)控分子,可以通過和SAP30的結(jié)合,增加經(jīng)輔抑制復(fù)合物Sin3A而發(fā)揮功能的HDACs酶的活性,從而使得結(jié)合HSV-1基因啟動子區(qū)域的組蛋白成分H3的第9、14位賴氨酸去乙�；缴险{(diào),其結(jié)果在一定程度上抑制了HSV-1相關(guān)基因的轉(zhuǎn)錄過程,并影響病毒的復(fù)制增殖。這一模型的建立,為我們更深入地理解HSV-1與宿主細(xì)胞相互作用通過細(xì)胞蛋白修飾酶系統(tǒng)來影響病毒基因轉(zhuǎn)錄調(diào)控,從而導(dǎo)致HSV-1感染細(xì)胞所致的多種病理學(xué)結(jié)果提供了實驗依據(jù)。
[Abstract]:Herpes simplex virus type I (HSV-1) is a pathogen with strong contagion and serious harm to human health. Human is the only host and there is no corresponding vaccine yet. As a complex structure, and in the process of gene transcription, the double chain DNA virus that shows clear cascade effect, HSV-1 in the process of interaction with cells. The analysis of the function of the early gene products caused by the HSV-1 virus binding receptor not only deepens the understanding of the interaction between HSV-1 and cells, but also helps to elucidate the mode of action of the unknown functional proteins in the cells and their molecular mechanisms. In this field, our previous study found that HS After the combination of V-1 with the surface receptor of human fibroblast KMB-17 strain, it can stimulate the difference in the level of cell gene expression. At the same time, we have made a more in-depth study by using mRNA differential display and protein 2D differentially induced cell related molecules, and screened some new genes from it. The HTRP of this study. Gene, HTRP (human transcription regulator protein), one of the genes (human transcription regulator protein) encoded by one of the unknown functions cloned in the above research work (AF442486), is a protein molecule associated with cell transcription regulation, showing SAP30 (mSin3 A) that is one of the components of the transcriptional assisted inhibition complex mSin3A. The interaction between Association Protein and SAP30 promotes the biological effects of HSV-1 infection on cell death.
This research focuses on the biological functions and molecular mechanisms of HTRP and SAP30 molecules in the interaction between cells and hosts, reveals the universal transcriptional inhibition effect of HTRP on the HSV-1 virus, and further confirms the synergistic effect of transcription inhibition between HTRP and SAP30, and probes into the interaction and transcriptional inhibition of both HTRP and SAP30. Close connection and its molecular mechanism. First, we use the cell environment that up-regulated the expression of HTRP to study its effect on the replication and proliferation of the virus. The results show that HTRP can inhibit the transcriptional activity of TK, gC gene in different stages of infection, and the inhibition is more obvious with the prolongation of time. Then, the up-regulated cells are expressed at the same time in HTRP and SAP30. In the environment, the synergistic effect of both TK and gC gene transcription inhibition is further confirmed. Research data on cell molecular biology have shown that SAP30 is one of the important structural components in the cooppressor complex of intracellular transcriptional regulation, which mediates the histone deacetylase (HI stone deacetyltransferases, HDAC). In combination with the function of the complex, our experiments showed that HTRP and SAP30 could inhibit the transcriptional inhibition of the enzyme gene by making the chromatin histone deacetylation pathway. In order to further verify the molecular mechanism of the synergistic inhibitory effect of HTRP and SAP30 on the virus gene, the HDACs inhibitor TSA was added to the double luciferase detection system. The experiment proved that the synergistic effect was realized through the changes in the activity of HDAC enzyme. At the same time, the use of chromatin immunoprecipitation (Chromatin Immunoprecipitation, ChIP) experiments showed that the activity of HDACs was changed by the combination of HTRP and SAP30, which made the HDACs enzyme enhanced the deacetylation level of the fourteenth and ninth bit lysine of the histone H3 molecule.
Based on the above results, the following model is proposed for the specific molecular biological mechanism of the cell protein molecule HTRP, which is specifically up-regulated by HSV-1 human fibroblasts. HTRP is a transcriptional regulator with a reactive up regulation of a cell in HSV-1 infection. It can be combined with SAP30 to increase the co inhibition compound. The activity of the function of Sin3A and the function of the HDACs enzyme makes the level of lysine deacetylation up-regulated in the 9,14 site of the histone component H3 of the promoter region of the HSV-1 gene, which inhibits the transcriptional process of the HSV-1 related genes and affects the replication and proliferation of the virus. Understanding the interaction of HSV-1 and host cells through the cell protein modified enzyme system affects the regulation of viral gene transcription, which leads to a variety of pathological results caused by HSV-1 infected cells.

【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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本文編號：1803407