本文關(guān)鍵詞：丙型肝炎病毒特異性樹突狀細(xì)胞胞外體的制備及免疫特性研究　出處：《第四軍醫(yī)大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 丙型肝炎病毒 樹突狀細(xì)胞 胞外體 免疫應(yīng)答

【摘要】： 丙型肝炎病毒(hepatitis C virus, HCV)是引起慢性肝炎的主要病原體,約80%HCV感染者發(fā)展為慢性肝炎,并與肝硬化和肝細(xì)胞肝癌的發(fā)生密切相關(guān),已成為嚴(yán)重的公共衛(wèi)生問題。然而,迄今尚缺乏有效的治療措施,疫苗的研制也步履維艱。樹突狀細(xì)胞(dendritic cell,DC)作為誘導(dǎo)固有免疫(innate immune)和適應(yīng)性免疫(adaptive immune)應(yīng)答的核心環(huán)節(jié),在機(jī)體抗病毒免疫應(yīng)答中發(fā)揮著重要作用,由DC分泌的胞外體(dendritic cell exosome, DC-Ex)富集MHC I/II類分子、協(xié)同刺激分子、熱休克蛋白70等多種生物活性分子于一身,作為細(xì)胞功能的使者,以“特洛伊木馬”的形式將免疫活化信息傳遞給其他免疫細(xì)胞,發(fā)揮特異性免疫效應(yīng)。因此,選擇性地為Ex載荷HCV抗原信息,理論上可以大大提高抗原的免疫原性,從而有效地誘導(dǎo)抗原特異性細(xì)胞免疫及體液免疫應(yīng)答,并建立抗病毒記憶反應(yīng),這為制備HCV疫苗提供了一個新的研究策略。 基于上述學(xué)術(shù)假設(shè),結(jié)合近年有關(guān)HCV研究所獲得的信息,本研究對DC-Ex的功能特性進(jìn)行以下兩方面的探討: 1、DC-Ex制備方法的初步建立及分析鑒定 ①DC-Ex制備方法的初步建立選用永生化的小鼠樹突狀細(xì)胞株DC2.4為研究對象,在尿酸(uric acid,UA)濃度為0.05mmol/L,0.1mmol/L,0.2mmol/L和0.3 mmol/L培養(yǎng)24 h條件下,收集細(xì)胞培養(yǎng)上清,捕獲ELISA法檢測Ex表面特異性標(biāo)志CD80、CD86的表達(dá)率,間接考量Ex產(chǎn)生的動力學(xué)水平,探索UA對DC分泌Ex的影響,為制備大量的較高純度的Ex做準(zhǔn)備;基于Ex的大小和密度,本研究對Ex傳統(tǒng)分離方法進(jìn)行改進(jìn),把超濾技術(shù)引入Ex的制備過程,即應(yīng)用中空纖維器去除細(xì)胞及細(xì)胞碎片、Amicon Ultra超濾離心管壓縮體積、密度梯度超離心純化獲得DC-Ex提取物。對照組則采用傳統(tǒng)的分級分離的制備方法,比較兩種方法的優(yōu)劣。 ②DC-Ex的分析鑒定應(yīng)用透射電鏡對DC-Ex提取物進(jìn)行形態(tài)學(xué)鑒定,十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(sodium dodecyl sulphte- polyacrylamide gel electrophoresis,SDS-PAGE)和Western blot進(jìn)行蛋白組成分析。 2、HCV特異性DC-Ex誘導(dǎo)的免疫應(yīng)答反應(yīng) 選用HCV核心蛋白C,體外刺激DC2.4細(xì)胞株,獲得具有HCV特異性的DC-Ex;把制備的C57BL/6小鼠脾細(xì)胞懸液與HCV特異性的DC-Ex共孵育。收集培養(yǎng)上清,酶聯(lián)免疫吸附試驗(yàn)(enzyme-linked immune assay,ELISA)檢測各組培養(yǎng)上清中IL-6、IL-12及IFN-γ的水平,探討DC-Ex的呈遞免疫活化信息、誘導(dǎo)抗原特異性免疫反應(yīng)的能力。 實(shí)驗(yàn)結(jié)果: 1、尿酸是DC分泌Ex的重要調(diào)節(jié)因素,以劑量依賴方式影響Ex的產(chǎn)量,濃度為0.1 mmol/L時,Ex的產(chǎn)量最高。 2、所建立的超濾離心法是一種簡便高效的制備Ex的實(shí)驗(yàn)方法,獲得的DC-Ex在電鏡下呈典型的杯狀,由脂質(zhì)雙層膜包繞的扁平半球體,直徑約60-90 nm,SDS-PAGE帶型和文獻(xiàn)報道相似,包含有主要的特征性標(biāo)志物分子MHC I,MHC II,CD80和CD86,與傳統(tǒng)分離法相比,超濾離心法耗時少,產(chǎn)量和純度較高。 3、HCV特異性DC-Ex可有效地將免疫活化信息呈遞給脾細(xì)胞,誘導(dǎo)脾細(xì)胞產(chǎn)生較高水平的IL-6、IL-12、IFN-γ。 初步實(shí)驗(yàn)結(jié)論: 在尿酸的誘導(dǎo)下,超濾離心法可成功獲得較高純度的Ex,所制備的HCV特異性DC-Ex能有效的將免疫活化信息呈遞給其他免疫細(xì)胞,誘導(dǎo)抗原特異免疫反應(yīng),有可能作為一種新的DC-Ex疫苗用于HCV感染的預(yù)防和治療。
[Abstract]:Hepatitis C virus (hepatitis C, virus, HCV) is a major pathogen of chronic hepatitis, about 80%HCV infection develop chronic hepatitis, and cirrhosis and hepatocellular carcinoma is closely related to the occurrence, has become a serious public health problem. However, there are still no effective treatment and vaccine development also said of an aged person. Dendritic cells (dendritic cell, DC (innate) as induction of innate immunity and adaptive immunity (Immune) adaptive immune) core link response, plays an important role in antiviral immune response, exosomes secreted by DC (dendritic cell exosome, DC-Ex) enrichment of MHC class I/II molecules, costimulatory molecules, heat shock protein 70 and other biologically active molecules in a cell function as a messenger, "Troy horse" in the form of immune activation information to other immune cells, play a specific Immunity effect. Therefore, the selective Ex HCV antigen load information theory can greatly improve the immunogenicity of the antigen, thus effectively induce antigen specific cellular immunity and humoral immune response, and the establishment of antiviral memory response, which provides a new strategy for the preparation of HCV vaccine.
Based on the above academic hypothesis, combined with the information obtained by the HCV Research Institute in recent years, this study explores the following two aspects of the functional characteristics of DC-Ex:
1, preliminary establishment and analysis and identification of DC-Ex preparation method
The preparation method of DC-Ex preliminary used to establish immortalized mouse dendritic cell line DC2.4 as the research object, the uric acid (uric acid, UA) concentration of 0.05mmol/L, 0.1mmol/L, H 24 0.2mmol/L and 0.3 mmol/L culture conditions, supernatants were collected to capture ELISA method to detect Ex specific surface markers CD80, CD86 expression rate level dynamics generated by Ex indirect considerations, to explore the effect of UA on Ex secretion of DC, preparing for the preparation of high purity of Ex; the size and density of Ex based on the study of improvement of Ex traditional separation methods, the preparation process of ultrafiltration technology into Ex, which is used for removing hollow fiber cells and cell debris, Amicon Ultra ultrafiltration centrifugal tube compression volume, density gradient ultracentrifugation purified extract of DC-Ex. The preparation method of the control group with conventional fractionation, compare the advantages and disadvantages of two methods.
(2) identification and identification of DC-Ex. Morphological identification of DC-Ex extracts was carried out by transmission electron microscope. Protein composition analysis was carried out by sodium dodecyl sulphte- polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot twelve sodium.
2, HCV specific DC-Ex induced immune response
Selection of HCV core protein C, in vitro stimulation of DC2.4 cells, with HCV specific DC-Ex; the preparation of C57BL/6 mouse spleen cell suspension with HCV specific DC-Ex were incubated. The supernatant was collected and enzyme-linked immunosorbent assay (enzyme-linked immune, assay, ELISA) were detected in the culture supernatant of IL-6, IL-12 IFN- and gamma levels of immune activation of DC-Ex presenting information, the ability to induce antigen-specific immune responses.
Experimental results:
1, uric acid is an important regulatory factor for the secretion of Ex by DC, which affects the yield of Ex in a dose dependent manner. When the concentration is 0.1 mmol/L, the production of Ex is the highest.
2, the centrifugal ultrafiltration is a simple and effective experimental method for the preparation of Ex, the DC-Ex is a typical goblet in the electron microscope by the lipid bilayer membrane bag flat hemispherical circle, the diameter of 60-90 nm, SDS-PAGE and similar reports in the literature, including the characteristic markers of molecular MHC I, MHC II, CD80 and CD86, compared with the traditional separation method, centrifugal ultrafiltration less time-consuming, yield and high purity.
3, HCV specific DC-Ex can effectively transmit immune activation information to spleen cells, inducing splenocytes to produce a high level of IL-6, IL-12, IFN- gamma.
Preliminary conclusion:
The uric acid induced by centrifugal ultrafiltration can be successfully obtained high purity Ex, DC-Ex prepared by HCV specific preparation can effectively present information to the immune activation of other immune cells, induce antigen specific immune response, may be used as a prevention and treatment of HCV infection for new DC-Ex vaccine.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392

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