丙型肝炎病毒特异性树突状细胞胞外体的制备及免疫特性研究

发布时间：2018-01-06 06:23

本文关键词：丙型肝炎病毒特异性树突状细胞胞外体的制备及免疫特性研究　出处：《第四军医大学》2008年硕士论文　论文类型：学位论文

【摘要】： 丙型肝炎病毒(hepatitis C virus, HCV)是引起慢性肝炎的主要病原体,约80%HCV感染者发展为慢性肝炎,并与肝硬化和肝细胞肝癌的发生密切相关,已成为严重的公共卫生问题。然而,迄今尚缺乏有效的治疗措施,疫苗的研制也步履维艰。树突状细胞(dendritic cell,DC)作为诱导固有免疫(innate immune)和适应性免疫(adaptive immune)应答的核心环节,在机体抗病毒免疫应答中发挥着重要作用,由DC分泌的胞外体(dendritic cell exosome, DC-Ex)富集MHC I/II类分子、协同刺激分子、热休克蛋白70等多种生物活性分子于一身,作为细胞功能的使者,以“特洛伊木马”的形式将免疫活化信息传递给其他免疫细胞,发挥特异性免疫效应。因此,选择性地为Ex载荷HCV抗原信息,理论上可以大大提高抗原的免疫原性,从而有效地诱导抗原特异性细胞免疫及体液免疫应答,并建立抗病毒记忆反应,这为制备HCV疫苗提供了一个新的研究策略。基于上述学术假设,结合近年有关HCV研究所获得的信息,本研究对DC-Ex的功能特性进行以下两方面的探讨: 1、DC-Ex制备方法的初步建立及分析鉴定 ①DC-Ex制备方法的初步建立选用永生化的小鼠树突状细胞株DC2.4为研究对象,在尿酸(uric acid,UA)浓度为0.05mmol/L,0.1mmol/L,0.2mmol/L和0.3 mmol/L培养24 h条件下,收集细胞培养上清,捕获ELISA法检测Ex表面特异性标志CD80、CD86的表达率,间接考量Ex产生的动力学水平,探索UA对DC分泌Ex的影响,为制备大量的较高纯度的Ex做准备;基于Ex的大小和密度,本研究对Ex传统分离方法进行改进,把超滤技术引入Ex的制备过程,即应用中空纤维器去除细胞及细胞碎片、Amicon Ultra超滤离心管压缩体积、密度梯度超离心纯化获得DC-Ex提取物。对照组则采用传统的分级分离的制备方法,比较两种方法的优劣。 ②DC-Ex的分析鉴定应用透射电镜对DC-Ex提取物进行形态学鉴定,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulphte- polyacrylamide gel electrophoresis,SDS-PAGE)和Western blot进行蛋白组成分析。 2、HCV特异性DC-Ex诱导的免疫应答反应选用HCV核心蛋白C,体外刺激DC2.4细胞株,获得具有HCV特异性的DC-Ex;把制备的C57BL/6小鼠脾细胞悬液与HCV特异性的DC-Ex共孵育。收集培养上清,酶联免疫吸附试验(enzyme-linked immune assay,ELISA)检测各组培养上清中IL-6、IL-12及IFN-γ的水平,探讨DC-Ex的呈递免疫活化信息、诱导抗原特异性免疫反应的能力。实验结果: 1、尿酸是DC分泌Ex的重要调节因素,以剂量依赖方式影响Ex的产量,浓度为0.1 mmol/L时,Ex的产量最高。 2、所建立的超滤离心法是一种简便高效的制备Ex的实验方法,获得的DC-Ex在电镜下呈典型的杯状,由脂质双层膜包绕的扁平半球体,直径约60-90 nm,SDS-PAGE带型和文献报道相似,包含有主要的特征性标志物分子MHC I,MHC II,CD80和CD86,与传统分离法相比,超滤离心法耗时少,产量和纯度较高。 3、HCV特异性DC-Ex可有效地将免疫活化信息呈递给脾细胞,诱导脾细胞产生较高水平的IL-6、IL-12、IFN-γ。初步实验结论: 在尿酸的诱导下,超滤离心法可成功获得较高纯度的Ex,所制备的HCV特异性DC-Ex能有效的将免疫活化信息呈递给其他免疫细胞,诱导抗原特异免疫反应,有可能作为一种新的DC-Ex疫苗用于HCV感染的预防和治疗。
[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.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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