负载EBV-LMP2基因人树突状细胞疫苗的制备及抗肿瘤免疫研究

发布时间：2018-01-03 18:11

本文关键词：负载EBV-LMP2基因人树突状细胞疫苗的制备及抗肿瘤免疫研究　出处：《汕头大学》2006年博士论文　论文类型：学位论文

【摘要】：目的 1．采用细胞因子组合GM-SCF、IL-4和TNF-α体外诱导人外周血单核细胞分化增殖为树突状细胞(dendritic cell，DC)，并以腺病毒为载体，将鼻咽癌(NPC)相关的Epstein-Barr病毒潜伏膜蛋白2(EBV-LMP2)转染DC，制备rAd-LMP2-DC疫苗。检测其生物学特性；体外诱导并测定细胞毒T淋巴细胞(cytotoxic T lymphocyte，CTL)活性；进而观察其对NPE荷瘤小鼠的免疫治疗作用，以及在hu-PBL-SCID小鼠体内对肿瘤的免疫防治效应。2．探索无血清条件下应用钙离子载体(CI)诱导成熟DC的方法。检测其生物学特性；体外诱导并测定CTL活性。方法 1．rAd-LMP2-DC疫苗的制备：采用细胞因子rhGM-CSF、rhIL-4和rhTNF-α组合诱导人外周血单核细胞向DC分化，，在重组腺病毒介导下将EBV-LMP2基因转染DC，制备rAd-LMP2-DC疫苗。动态观察细胞形态学变化，扫描电镜观察成熟DC的形态学特征；FACS检测DC表型CD80、CD86、CD83及LMP2阳性表达率。2．体外抗肿瘤实验：采用混合淋巴细胞反应，用rAd-LMP2-DC刺激T细胞诱导生成LMP2特异性CTL，并用MTT法检测CTL对NPC肿瘤细胞CNE-2的杀伤活性，观察DC刺激次数对CTL杀伤活性的影响，以及FACS检测CTL中CD3~+、CD4~+、CD8~+的组成。3．体内抗肿瘤实验：选用SCID小鼠为实验动物。(1)建立SCID小鼠-人淋巴细胞嵌合模型：人外周血T
[Abstract]:Objective 1. To induce the proliferation of human peripheral blood monocytes into dendritic cell dendritic cell in vitro by cytokine combination of GM-SCF IL-4 and TNF- 伪. Epstein-Barr virus latent membrane protein 2EBV-LMP2 was transfected into DC by adenovirus. RAd-LMP2-DC vaccine was prepared and its biological characteristics were tested. Cytotoxic T lymphocytes (CTL) were induced and measured in vitro. Then the immunotherapy of NPE bearing mice was observed. 2. To explore the method of inducing mature DC by using calcium ion carrier in serum free condition, and to detect its biological characteristics; 2. To investigate the immune effect of hu-PBL-SCID mice on tumor prevention and treatment. 2. To explore the method of inducing mature DC by using calcium ion carrier in serum free condition. CTL activity was induced and measured in vitro. Methods 1. Preparation of rAd-LMP2-DC vaccine: cytokine rhGM-CSF was used. The combination of rhIL-4 and rhTNF- 伪 induces the differentiation of human peripheral blood monocytes into DC, and EBV-LMP2 gene is transfected into DC mediated by recombinant adenovirus. RAd-LMP2-DC vaccine was prepared. Morphological changes of cells were observed dynamically and morphological characteristics of mature DC were observed by scanning electron microscope (SEM). FACS was used to detect the positive expression rate of CD80, CD86, CD83 and LMP2. 2. Anti-tumor experiment in vitro: mixed lymphocyte reaction was used. RAd-LMP2-DC was used to stimulate T cells to induce the formation of LMP2 specific CTLs and MTT assay was used to detect the cytotoxicity of CTL to CNE-2 of NPC tumor cells. The effects of DC stimulation times on the killing activity of CTL and the detection of CD3 ~ + CD4 ~ in CTL by FACS were observed. In vivo anti-tumor experiment: SCID mice were selected as experimental animals to establish SCID murine-human lymphocyte chimeric model: human peripheral blood T
【学位授予单位】：汕头大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392;R73-36

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