本文關(guān)鍵詞：慢性病毒感染中CD8T細(xì)胞功能耗竭的分子機(jī)制　出處：《上海交通大學(xué)》2012年博士論文　論文類型：學(xué)位論文

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【摘要】：背景和目的：慢性病毒感染后，CD8T細(xì)胞增殖分化成為病毒特異性的效應(yīng)CD8T細(xì)胞，由于慢性病毒持續(xù)存在，效應(yīng)CD8T細(xì)胞免疫功能逐漸降低，增殖能力下降，細(xì)胞存活率下降，并且不能分化形成記憶性CD8T細(xì)胞，這一現(xiàn)象已經(jīng)被學(xué)術(shù)界廣泛研究發(fā)現(xiàn)，并且稱此為T(mén)細(xì)胞功能耗竭,大部分發(fā)生在慢性感染和腫瘤中。目前關(guān)于CD8T細(xì)胞功能耗竭的機(jī)制尚不完全清楚。我們的前期研究結(jié)果顯示，表觀遺傳修飾在記憶性CD8T細(xì)胞的分化形成以及功能發(fā)揮中具有重要作用。因此本研究中，我們將首次從表觀遺傳修飾角度探討慢性病毒感染中CD8T細(xì)胞功能耗竭的分子調(diào)控機(jī)制，重點(diǎn)是組蛋白乙�；{(diào)控與病毒慢性感染中功能耗竭性CD8T細(xì)胞的關(guān)系，從而以此為理論基礎(chǔ)進(jìn)一步研究延緩或者逆轉(zhuǎn)慢性病毒感染后CD8T細(xì)胞功能的耗竭。 方法：利用淋巴細(xì)胞行脈絡(luò)叢腦膜炎病毒(lymphocytic choriomeningitisvirus-LCMV)不同病毒株感染小鼠建立小鼠感染模型：急性病毒株LCMV-Arm（Arm）感染小鼠建立急性感染，LCMV-clone13（Cl-13）感染小鼠建立慢性感染。通過(guò)與Arm急性病毒感染比較，分析Cl-13慢性感染過(guò)程中，病毒特異性CD8T細(xì)胞組蛋白乙�；谋磉_(dá)特點(diǎn)、功能意義及與疾病進(jìn)展的關(guān)系，從表觀遺傳學(xué)角度分析慢性感染中抗原特異性CD8T細(xì)胞免疫功能耗竭的分子機(jī)制。應(yīng)用四聚體染色技術(shù)檢測(cè)表位特異性CD8T細(xì)胞的頻率；流式細(xì)胞儀檢測(cè)病毒特異性CD8T細(xì)胞的表型特征；體外多肽刺激淋巴細(xì)胞，流式細(xì)胞儀檢測(cè)CD8T細(xì)胞產(chǎn)生細(xì)胞因子的能力；流式細(xì)胞染色以及染色質(zhì)免疫共沉淀(chromatinimmunoprecipitation, ChIP）實(shí)驗(yàn)研究CD8T細(xì)胞組蛋白乙�；磉_(dá)水平；體內(nèi)和體外實(shí)驗(yàn)探討組蛋白乙�；敢种苿┍焖徕c（valproic acid sodium,VPA）是否可以逆轉(zhuǎn)耗竭性CD8T細(xì)胞的免疫功能；流式細(xì)胞染色和實(shí)時(shí)定量PCR檢測(cè)VPA處理后CD8T細(xì)胞的功能狀態(tài)；細(xì)胞過(guò)繼轉(zhuǎn)移實(shí)驗(yàn)研究VPA體外處理后的功能耗竭性CD8T細(xì)胞是否可以在體內(nèi)存活并且發(fā)揮更強(qiáng)的免疫功能。 結(jié)果：1)與Arm急性致病病毒株比較，Cl-13慢性病毒株感染小鼠后，病原特異性CD8T細(xì)胞免疫功能逐漸降低，分泌細(xì)胞因子IFN-γ, TNF-α以及IL-2能力下降，并最終進(jìn)入耗竭狀態(tài)。2) Cl-13感染中，流式細(xì)胞儀染色分析顯示，，病毒特異性CD8T細(xì)胞組蛋白乙�；皆诟腥境跗诟弑磉_(dá)，隨著感染的進(jìn)程，逐漸低表達(dá)。ChIP結(jié)果顯示，在慢性病毒感染初期，ifng-promoter和ifng-enhancer兩基因位點(diǎn)結(jié)合更多乙�；慕M蛋白；在功能耗竭期，兩基因位點(diǎn)結(jié)合乙�；慕M蛋白水平顯著降低。3) Cl-13感染中，不僅病毒特異性CD8T細(xì)胞功能耗竭，整體CD8T細(xì)胞免疫功能也下降，表現(xiàn)為分泌細(xì)胞因子的能力降低，并且整體CD8T細(xì)胞組蛋白乙�；揭步档�。4)體外VPA處理耗竭性CD8T細(xì)胞后，其組蛋白乙�；缴�，分泌IFN-γ等細(xì)胞因子的能力提高。5)體外VPA處理后的耗竭性CD8T細(xì)胞過(guò)繼轉(zhuǎn)移入正常小鼠體內(nèi)后，存活率升高，并且分泌細(xì)胞因子能力升高，表明這群細(xì)胞免疫功能增強(qiáng)，成為具有功能的記憶性CD8T細(xì)胞。6)體內(nèi)VPA治療Cl-13病毒感染后小鼠，病毒特異性CD8T細(xì)胞組蛋白乙�；缴�，耗竭性CD8T細(xì)胞延緩形成。7)體內(nèi)或者體外VPA處理耗竭性CD8T細(xì)胞后，ChIP實(shí)驗(yàn)結(jié)果顯示，ifng-promoter和ifng-enhancer兩基因位點(diǎn)結(jié)合乙�；慕M蛋白水平顯著升高。 結(jié)論：Cl-13慢性病毒株感染后，CD8T細(xì)胞免疫功能漸進(jìn)性耗竭；Cl-13感染過(guò)程中CD8T細(xì)胞功能缺陷與其不能進(jìn)行必需的組蛋白乙�；厮苡嘘P(guān)；慢性感染過(guò)程中CD8T細(xì)胞功能缺陷可以通過(guò)增加組蛋白乙�；靡阅孓D(zhuǎn)。本研究首次從表觀遺傳學(xué)角度闡明了病毒慢性感染后CD8T細(xì)胞功能耗竭的機(jī)制，從而為病毒慢性感染的免疫調(diào)節(jié)治療提供理論基礎(chǔ)與實(shí)驗(yàn)依據(jù)。
[Abstract]:Background and objective: chronic virus infection, proliferation and differentiation of CD8T cells into virus specific CD8T cells that are due to chronic virus persistence, CD8T cell immune function effect is gradually reduced, the proliferation ability decreased, cell survival rate decreased, and cannot differentiate into memory CD8T cells, this phenomenon has been widespread academic research found, and called for the function of T cell depletion, mostly in chronic infections and tumors. The mechanism of CD8T cell depletion is not completely understood. Our preliminary results show that epigenetic modification plays an important role in the formation of differentiation of memory CD8T cells and function in the study. We will, for the first time from the perspective of epigenetic modification to explore the molecular mechanism of chronic viral infection in CD8T cell depletion, especially histone acetylation and disease control Based on the relationship between functional depletion CD8T cells in chronic infection, we further study the depletion of CD8T cell function after delaying or reversing chronic viral infection.
Methods: for lymphocyte choriomeningitis virus (lymphocytic choriomeningitisvirus-LCMV) with different strains of mice infected mice to establish infection model: acute virus strain LCMV-Arm (Arm) infection in mice acute infection, LCMV-clone13 (Cl-13) infection in mice to establish chronic infections. By comparison with Arm acute viral infection, analysis during chronic Cl-13 infection, virus specific CD8T cell histone acetylation expression characteristics, functional significance and the relationship with disease progression, from the perspective of epigenetics analysis of molecular mechanisms of chronic infection of antigen-specific CD8T cell immune function exhaustion. Four application of dimer staining technique for the detection of epitope specific CD8T cell phenotype frequency; detection of virus specific CD8T flow cytometry; in vitro peptide stimulated lymphocytes, cytokine production of CD8T cells were detected by flow cytometry Capacity; flow cytometry staining and chromatin immunoprecipitation (chromatinimmunoprecipitation, ChIP) CD8T cells histone acetylation levels; in vivo and in vitro experiments of histone deacetylase inhibitor valproic acid (valproic acid sodium, VPA) could reverse immune function of CD8T cell exhaustion; the functional status of CD8T cells VPA processing flow cytometry staining and real-time PCR cells; adoptive transfer function is exhausted CD8T cell VPA in vitro after treatment and can play a stronger immune function in vivo.
Results: 1) compared with Arm acute pathogenic strains, Cl-13 strains of mice infected with chronic virus, pathogen specific CD8T cell immune function decreased, secretion of cytokines IFN-, TNF- alpha and IL-2 decreased, and eventually into the exhaustion of Cl-13 infection,.2) staining and flow cytometry analysis showed that virus specific CD8T cells with high expression level of histone acetylation in the early stage of infection, with the infection process, has low expression of.ChIP showed that in the early stage of chronic viral infection, ifng-promoter and ifng-enhancer genes with more acetylation of histone; in the functional exhaustion period, genes with acetylated histones were significantly reduced.3) Cl-13 infection, not only virus specific CD8T cell function, CD8T cell immune function overall also decreased, decreased secretion of cytokine, and whole cell CD8T Histone acetylation levels also decreased.4) in vitro VPA depletion of CD8T cells, increased the level of histone acetylation, ability to secrete IFN- and other cytokines in vitro after VPA treatment increased.5) depletion of adoptive transfer of CD8T cells into normal mice, the survival rate increased, and the ability to increase the secretion of cytokines, showed that these cells enhanced immune function, as has the function of memory CD8T cells in VPA.6) for the treatment of Cl-13 virus infection in mice, increased virus specific CD8T cell histone acetylation, depletion of CD8T cells delayed the formation of.7) in vivo or in vitro depletion of CD8T cells after VPA treatment, ChIP results showed that the ifng-promoter and ifng-enhancer genes with acetylated histone levels increased significantly.
Conclusion: Cl-13 chronic virus infection, CD8T cell immune function, progressive exhaustion; the process of Cl-13 infection CD8T cell function defect and not histone acetylation remodeling required; CD8T cell function defect during chronic infection can be reversed by increasing histone acetylation. This is the first study from the perspective of epigenetics the chronic virus infection after CD8T cell depletion mechanism, which is the chronic virus infection immune regulation and provide a theoretical basis and experimental basis for the treatment.

【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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相關(guān)期刊論文 前1條

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