【摘要】：慢性乙型肝炎仍是嚴(yán)重危害人類健康的疾病,現(xiàn)今全球大約有3.5億人感染乙肝病毒(hepatitis B virus,HBV)。目前采取的治療方法,均不能徹底清除病毒而使病毒持續(xù)存在于肝細(xì)胞內(nèi),造成感染慢性化,并可繼發(fā)肝硬化、肝細(xì)胞癌。機(jī)體清除HBV感染起關(guān)鍵作用的是HBV特異性細(xì)胞毒T淋巴細(xì)胞(cytotoxic T lymphocyte,CTL)。泛素-蛋白酶體系統(tǒng)(ubiquitin-proteasome system,UPS)是一種高度選擇性的蛋白降解系統(tǒng),它廣泛存在于真核細(xì)胞內(nèi),并依靠ATP酶而起作用,對(duì)維持細(xì)胞乃至整個(gè)生物體的正常生理功能具有重要的作用。泛素化的抗原蛋白被蛋白酶體復(fù)合物識(shí)別后被降解為若干小肽段,與主要組織相容性復(fù)合體-I(major histocompatibility complex,MHC)類分子結(jié)合后被抗原提呈細(xì)胞識(shí)別,誘導(dǎo)特異性CTL反應(yīng)。目前有許多研究表明,泛素化的抗原進(jìn)入細(xì)胞后可被UPS系統(tǒng)有效降解及提呈,從而增強(qiáng)抗原誘導(dǎo)的免疫應(yīng)答。正常情況下,由于膜屏障的存在,蛋白、多肽等很難進(jìn)入細(xì)胞內(nèi),某些蛋白具有很強(qiáng)的不依賴于受體的跨膜活性,這類短肽被稱為細(xì)胞穿透肽(cell penetrating peptide,CPP)。目前應(yīng)用最為廣泛的細(xì)胞穿透肽是HIV-Tat(human immunodeficiency virus transactivator oftranscription,HIV-Tat)蛋白。PTD(protein transduction domain)是Tat蛋白行使跨膜功能的核心片段,包含該區(qū)段的蛋白具有穿透細(xì)胞膜的功能。胞漿轉(zhuǎn)導(dǎo)肽(cytoplasmic transduction peptide,CTP)為PTD的衍生體,是一種新型的轉(zhuǎn)導(dǎo)肽系統(tǒng),它去除了核定位信號(hào)而能攜帶蛋白類大分子穿越細(xì)胞膜并專一性定位于胞漿中的。本研究通過構(gòu)建Ub-HBc Ag-CTP融合基因表達(dá)質(zhì)粒,并進(jìn)行蛋白的表達(dá)及純化,利用Ub-HBc Ag-CTP融合蛋白體外刺激樹突狀細(xì)胞(dendritic cell,DC)成熟并誘導(dǎo)特異性CTL,體內(nèi)免疫BALB/c小鼠,對(duì)Ub-HBc Ag-CTP融合蛋白誘導(dǎo)特異性CTL的免疫功能進(jìn)行了探討。實(shí)驗(yàn)共分三部分:(1)Ub-HBcAg-CTP融合蛋白的表達(dá)、純化及其體外誘導(dǎo)小鼠髓源性樹突狀細(xì)胞成熟的研究;(2)Ub-HBc Ag-CTP融合蛋白促進(jìn)T淋巴細(xì)胞增殖,誘導(dǎo)特異性ctl反應(yīng)的體外研究;(3)ub-hbcag-ctp融合蛋白免疫balb/c小鼠誘導(dǎo)特異性ctl反應(yīng)的體內(nèi)研究。第一部分Ub-HBcAg-CTP融合蛋白的表達(dá)、純化及其體外誘導(dǎo)小鼠髓源性樹突狀細(xì)胞成熟的研究目的:構(gòu)建Ub-HBcAg-CTP融合基因的原核表達(dá)載體,并進(jìn)行蛋白的表達(dá)及其純化,體外觀察其對(duì)誘導(dǎo)小鼠髓源性樹突狀細(xì)胞成熟的作用。方法:以質(zhì)粒pcDNA3.1(-)-Ub-HBcAg中的Ub-HBc Ag融合基因?yàn)槟０逶O(shè)計(jì)引物,上游引物帶有CTP基因序列,進(jìn)行PCR擴(kuò)增,PCR產(chǎn)物克隆到原核表達(dá)質(zhì)粒pMAL-c2X中,菌落PCR陽(yáng)性克隆測(cè)序,鑒定正確的質(zhì)粒轉(zhuǎn)化大腸桿菌BL21(DE3),并進(jìn)行蛋白純化及Western blotting鑒定。同時(shí)構(gòu)建并表達(dá)對(duì)照組蛋白HBcAg-CTP及Ub-HBcAg。體外分離培養(yǎng)近交系BALB/c小鼠髓源性樹突狀細(xì)胞,加重組白細(xì)胞介素-4(interleukin,IL-4)及粒細(xì)胞-巨噬細(xì)胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)。樹突狀細(xì)胞培養(yǎng)至第5天時(shí)加入U(xiǎn)b-HBc Ag-CTP、HBc Ag-CTP、Ub-HBcAg及HBc Ag誘導(dǎo)DC成熟。激光共聚焦顯微鏡觀察免疫熒光在細(xì)胞中的分布及定位,并對(duì)熒光強(qiáng)度進(jìn)行定量分析,流式細(xì)胞術(shù)檢測(cè)樹突狀細(xì)胞表面分子的表達(dá),并以酶聯(lián)免疫吸附法(ELISA)檢測(cè)樹突狀細(xì)胞的上清液中細(xì)胞因子IL-12p70的含量。結(jié)果:PCR擴(kuò)增分別得到820bp、590bp及780bp大小的條帶,分別為Ub-HBcAg-CTP、HBc Ag-CTP及Ub-HBcAg融合基因。將其克隆至表達(dá)質(zhì)粒pMAL-c2X中,陽(yáng)性克隆菌落測(cè)序正確,并在DE3中獲得誘導(dǎo)表達(dá)。Western blotting鑒定分別為Ub-HBcAg-CTP、HBc Ag-CTP及Ub-HBc Ag融合蛋白。體外成功誘導(dǎo)培養(yǎng)并鑒定小鼠骨髓源性DC,免疫熒光法證實(shí)Ub-HBcAg-CTP能夠穿透DC膜進(jìn)入細(xì)胞質(zhì),而不能進(jìn)入細(xì)胞核;Ub-HBcAg-CTP能明顯上調(diào)DC表面分子CD80、CD83、CD86及MHC-I類分子的表達(dá);Ub-HBc Ag-CTP組誘導(dǎo)DC分泌的IL-12p70水平明顯高于對(duì)照組。結(jié)論:成功構(gòu)建了Ub-HBcAg-CTP及對(duì)照融合表達(dá)質(zhì)粒并誘導(dǎo)表達(dá),Ub-HBcAg-CTP具有穿透樹突狀細(xì)胞膜并定位于胞漿的能力,明顯增強(qiáng)DC表面共刺激分子的表達(dá)及促進(jìn)DC的成熟及分化,明顯增強(qiáng)DC分泌IL-12p70等細(xì)胞因子的能力。第二部分Ub-HBc Ag-CTP融合蛋白促進(jìn)T淋巴細(xì)胞增殖,誘導(dǎo)特異性CTL反應(yīng)的體外研究目的:探討經(jīng)Ub-HBc Ag-CTP融合蛋白致敏的DC體外增強(qiáng)T淋巴細(xì)胞增殖能力及誘導(dǎo)特異性細(xì)胞毒T淋巴細(xì)胞(CTLs)的作用。方法:體外分離培養(yǎng)小鼠髓源性DC,不同組融合蛋白加入樹突狀細(xì)胞中誘導(dǎo)其成熟及分化后,與分離培養(yǎng)的小鼠T淋巴細(xì)胞共同培養(yǎng),ELISA法檢測(cè)T細(xì)胞上清液中干擾素(interferon,IFN)-γ、IL-2、IL-4及IL-10的分泌水平,流式細(xì)胞儀檢測(cè)胞內(nèi)細(xì)胞因子水平,細(xì)胞計(jì)數(shù)試劑盒-8(CCK-8)試劑盒檢測(cè)T淋巴細(xì)胞增殖反應(yīng),乳酸脫氫酶(LDH)釋放試驗(yàn)檢測(cè)特異性CTL活性。結(jié)果:Ub-HBc Ag-CTP融合蛋白可以明顯上調(diào)細(xì)胞因子IFN-γ和IL-2的水平;流式細(xì)胞儀檢測(cè)的由Ub-HBc Ag-CTP融合蛋白所誘導(dǎo)的CTL水平明顯高于對(duì)照組及空白組;Ub-HBc Ag-CTP誘導(dǎo)樹突狀細(xì)胞刺激T細(xì)胞增殖能力明顯高于對(duì)照組;Ub-HBc Ag-CTP融合蛋白組誘導(dǎo)的CTL與對(duì)照組相比,具有明顯的特異性殺傷作用。結(jié)論:經(jīng)Ub-HBc Ag-CTP融合蛋白誘導(dǎo)成熟的DC能明顯刺激Th1型細(xì)胞因子的分泌,增強(qiáng)T淋巴細(xì)胞增殖能力,明顯增加CTLs的表達(dá)并增強(qiáng)特異性CTL活性。第三部分Ub-HBc Ag-CTP融合蛋白免疫BALB/c小鼠誘導(dǎo)特異性CTL反應(yīng)的體內(nèi)研究目的:探討Ub-HBc Ag-CTP融合蛋白在BALB/c小鼠體內(nèi)有效誘導(dǎo)HBV特異性CTL反應(yīng),并初步探討Ub-HBc Ag-CTP融合蛋白誘導(dǎo)CTL的機(jī)制,研究Ub-HBc Ag-CTP融合蛋白誘導(dǎo)CTL與JAK/STAT信號(hào)通路的關(guān)系。方法:BALB/c小鼠隨機(jī)分為實(shí)驗(yàn)組Ub-HBc Ag-CTP(50μg),對(duì)照組HBc Ag-CTP(50μg)、Ub-HBc Ag(50μg)、HBc Ag(50μg)及空白組(100μl生理鹽水),經(jīng)肌肉免疫小鼠,每周一次,共3次。最后一次免疫后1周,收集小鼠T淋巴細(xì)胞,ELISA法檢測(cè)T淋巴細(xì)胞分泌細(xì)胞因子;流式細(xì)胞儀檢測(cè)T淋巴細(xì)胞內(nèi)的細(xì)胞因子;酶聯(lián)免疫斑點(diǎn)(ELISPOT)法檢測(cè)特異性T淋巴細(xì)胞;CCK-8試劑盒檢測(cè)T淋巴細(xì)胞增殖活性;LDH釋放試驗(yàn)檢測(cè)特異性CTL活性;Western blotting法檢測(cè)小鼠T淋巴細(xì)胞JAK/STAT信號(hào)通路中各信號(hào)分子的表達(dá)水平。結(jié)果:Ub-HBc Ag-CTP融合蛋白能有效刺激小鼠T淋巴細(xì)胞分泌Th1型細(xì)胞因子;流式細(xì)胞儀及ELISPOT法檢測(cè)該融合蛋白誘導(dǎo)的CTL水平明顯高于其他組;且該融合蛋白誘導(dǎo)的T淋巴細(xì)胞增殖活性和CTL活性明顯高于對(duì)照組及空白組;Ub-HBc Ag-CTP融合蛋白可以明顯上調(diào)T淋巴細(xì)胞內(nèi)Jak2,Tyk2,STAT1及STAT4的表達(dá)水平,而對(duì)Jak1,Jak3及STAT6的表達(dá)無統(tǒng)計(jì)學(xué)意義。結(jié)論:Ub-HBc Ag-CTP融合蛋白免疫BALB/c小鼠后,能有效刺激T淋巴細(xì)胞分泌Th1型細(xì)胞因子及增加CTLs的表達(dá),并能提高T淋巴細(xì)胞增殖活性及CTL活性,以上免疫效應(yīng)可能與JAK/STAT信號(hào)通路的活化有關(guān)。
[Abstract]:Chronic hepatitis B is still a serious disease endangering human health. There are about 350 million people infected with hepatitis B virus (HBV) in the world today. Current treatment methods can not completely eliminate the virus and make it persist in the liver cells, causing chronic infection, and secondary cirrhosis, hepatocellular carcinoma. Infection plays a key role in HBV-specific cytotoxic T lymphocyte (CTL). Ubiquitin-proteasome system (UPS) is a highly selective protein degradation system, which exists widely in eukaryotic cells, and depends on ATPase to maintain the integrity of cells and even the whole organism. Ubiquitinized antigen proteins are recognized by proteasome complexes and degraded into small peptides. They are recognized by antigen presenting cells after binding to major histocompatibility complex (MHC) molecules and induce specific CTL reactions. Normally, because of the existence of membrane barrier, proteins, peptides, etc. are difficult to enter the cell. Some proteins have strong transmembrane activity independent of receptors. These short peptides are called cell penetrating peptides. The most widely used cell penetrating peptide is HIV-Tat protein. PTD (protein transduction domain) is the core fragment of the transmembrane function of Tat protein. The protein containing this fragment has the function of penetrating cell membrane. Smc transduction peptide (CTP), a derivative of PTD, is a new type of peptide transduction system. It removes nuclear localization signal and carries protein macromolecules across cell membrane and specifically locates in cytoplasm. In this study, we constructed Ub-HBc Ag-CTP fusion gene expression plasmid, expressed and purified the protein, using Ub-HBc Ag-C. TP fusion protein stimulated dendritic cell (DC) maturation in vitro and induced specific CTL. BALB / c mice were immunized in vivo. The immune function of specific CTL induced by Ub-HBcAg-CTP fusion protein was investigated. The experiment was divided into three parts: (1) Expression, purification and induction of myeloid dendritic cells in vitro by Ub-HBcAg-CTP fusion protein. Studies on cell maturation; (2) Ub-HBcAg-CTP fusion protein promotes T lymphocyte proliferation and induces specific CTL reaction in vitro; (3) in vivo study on the induction of specific CTL reaction in balb/c mice immunized with ub-hbcag-ctp fusion protein. Part I Expression, purification and in vitro induction of mouse myeloid dendritic cells by Ub-HBcAg-CTP fusion protein Objective: To construct the prokaryotic expression vector of Ub-HBcAg-CTP fusion gene, express and purify the protein, and observe its effect on inducing maturation of murine myeloid dendritic cells in vitro. Colony PCR positive cloning and sequencing were carried out to identify the correct plasmid transformed into Escherichia coli BL21 (DE3) and to purify the protein and identify it by Western blotting. The control group proteins HBcAg-CTP and Ub-HBcAg were constructed and expressed in vitro. Dendritic cells, interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in aggravating group. DC maturation was induced by adding Ub-HBc Ag-CTP, HBc Ag-CTP, Ub-HBcAg and HBc Ag on the 5th day of culture. The distribution and localization of light in the cells were analyzed quantitatively. The expression of surface molecules of dendritic cells was detected by flow cytometry. The content of cytokine IL-12p70 in the supernatant of dendritic cells was detected by enzyme-linked immunosorbent assay (ELISA). Results: A band of 820 bp, 590 BP and 780 BP was amplified by PCR, respectively. Ub-HBcAg-CTP, HBc Ag-CTP and Ub-HBcAg fusion genes were cloned into the expression plasmid pMAL-c2X. The positive clones were sequenced correctly and expressed in DE3. The fusion proteins of Ub-HBcAg-CTP, HBc Ag-CTP and Ub-HBc Ag were identified by Western blotting. It was confirmed that Ub-HBcAg-CTP could penetrate DC membrane into cytoplasm but not into nucleus; Ub-HBcAg-CTP could significantly up-regulate the expression of CD80, CD83, CD86 and MHC-I molecules on DC surface; and the level of IL-12p70 secreted by Ub-HBcAg-CTP group was significantly higher than that of control group. Ub-HBcAg-CTP has the ability to penetrate the dendritic cell membrane and localize in the cytoplasm. It can significantly enhance the expression of costimulatory molecules on the surface of DC, promote the maturation and differentiation of DC, and enhance the ability of DC to secrete IL-12p70 and other cytokines. Part II Ub-HBcAg-CTP fusion protein promotes the proliferation of T lymphocytes and induces specific CTL reaction. Objective: To investigate the effect of DC sensitized by Ub-HBc Ag-CTP fusion protein on enhancing T lymphocyte proliferation and inducing specific cytotoxic T lymphocyte (CTLs) in vitro. Methods: Mice myelogenous DC was isolated and cultured in vitro. Different groups of fusion proteins were added to dendritic cells to induce its maturation and differentiation, then T lymphocyte was isolated and cultured. The secretion levels of interferon-gamma, IL-2, IL-4 and IL-10 in the supernatant of T cells were detected by ELISA, cytokine levels were detected by flow cytometry, T lymphocyte proliferation was detected by CCK-8 kit, and specific CTL activity was detected by lactate dehydrogenase (LDH) release assay. B-HBc Ag-CTP fusion protein can significantly up-regulate the levels of cytokines IFN-gamma and IL-2; flow cytometry showed that the level of CTL induced by Ub-HBc Ag-CTP fusion protein was significantly higher than that of control group and blank group; Ub-HBc Ag-CTP induced dendritic cells proliferation was significantly higher than that of control group; Ub-HBc Ag-CTP induced dendritic cells proliferation was significantly higher than that of Ub-HBc Ag-CTP fusion protein group; Conclusion: Mature DC induced by Ub-HBc Ag-CTP fusion protein can significantly stimulate the secretion of Th1 cytokines, enhance the proliferation of T lymphocytes, significantly increase the expression of CTLs and enhance the specific CTL activity. Part III Ub-HBc Ag-CTP fusion protein immunized BALB/c mice. Objective: To investigate the effect of Ub-HBc Ag-CTP fusion protein on inducing specific CTL reaction in BALB/c mice and the mechanism of inducing CTL by Ub-HBc Ag-CTP fusion protein. Methods: BALB/c mice were randomly divided into two groups. Ub-HBc Ag-CTP (50 ug), HBc Ag-CTP (50 ug), Ub-HBc Ag (50 ug), HBc Ag (50 ug), HBc Ag (50 ug) and blank group (100 UG normal saline) were immunized to mice once a week for three times. Cytokines; Enzyme-linked immunodot assay (ELISPOT) to detect specific T lymphocytes; CCK-8 kit to detect T lymphocyte proliferation activity; LDH release assay to detect specific CTL activity; Western blotting to detect the expression of signal molecules in mouse T lymphocyte JAK/STAT signaling pathway. Results: Ub-HBc Ag-CTP fusion protein was effective. The CTL level induced by the fusion protein was significantly higher than that of other groups, and the proliferation and CTL activity of T lymphocytes induced by the fusion protein were significantly higher than those of control group and blank group; Ub-HBc Ag-CTP fusion protein could significantly up-regulate Jak in T lymphocytes. Conclusion: Ub-HBc Ag-CTP fusion protein can effectively stimulate T lymphocytes to secrete Th1 cytokines and increase the expression of CTLs, and can increase the proliferation and CTL activity of T lymphocytes, which may be related to JAK/ST. Activation of AT signaling pathway is involved.
【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】：2015
【分類號(hào)】：R512.62

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