本文關鍵詞：負載EBV-LMP2基因人樹突狀細胞疫苗的制備及抗腫瘤免疫研究　出處：《汕頭大學》2006年博士論文　論文類型：學位論文

  更多相關文章： 樹突狀細胞 腺病毒載體 LMP2基因 細胞毒性T細胞 腫瘤疫苗 鈣離子載體

【摘要】：目的 1．采用細胞因子組合GM-SCF、IL-4和TNF-α體外誘導人外周血單核細胞分化增殖為樹突狀細胞(dendritic cell，DC)，并以腺病毒為載體，將鼻咽癌(NPC)相關的Epstein-Barr病毒潛伏膜蛋白2(EBV-LMP2)轉(zhuǎn)染DC，制備rAd-LMP2-DC疫苗。檢測其生物學特性；體外誘導并測定細胞毒T淋巴細胞(cytotoxic T lymphocyte，CTL)活性；進而觀察其對NPE荷瘤小鼠的免疫治療作用，以及在hu-PBL-SCID小鼠體內(nèi)對腫瘤的免疫防治效應。2．探索無血清條件下應用鈣離子載體(CI)誘導成熟DC的方法。檢測其生物學特性；體外誘導并測定CTL活性。 方法 1．rAd-LMP2-DC疫苗的制備：采用細胞因子rhGM-CSF、rhIL-4和rhTNF-α組合誘導人外周血單核細胞向DC分化，，在重組腺病毒介導下將EBV-LMP2基因轉(zhuǎn)染DC，制備rAd-LMP2-DC疫苗。動態(tài)觀察細胞形態(tài)學變化，掃描電鏡觀察成熟DC的形態(tài)學特征；FACS檢測DC表型CD80、CD86、CD83及LMP2陽性表達率。2．體外抗腫瘤實驗：采用混合淋巴細胞反應，用rAd-LMP2-DC刺激T細胞誘導生成LMP2特異性CTL，并用MTT法檢測CTL對NPC腫瘤細胞CNE-2的殺傷活性，觀察DC刺激次數(shù)對CTL殺傷活性的影響，以及FACS檢測CTL中CD3~+、CD4~+、CD8~+的組成。3．體內(nèi)抗腫瘤實驗：選用SCID小鼠為實驗動物。(1)建立SCID小鼠-人淋巴細胞嵌合模型：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- 偽. 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- 偽 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
【學位授予單位】：汕頭大學
【學位級別】：博士
【學位授予年份】：2006
【分類號】：R392;R73-36

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