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目的:近年来,树突状细胞参与调节作用的研究较多。实验拟验证重组腺相关病毒(rAAV)介导癌胚抗原基因转染树突状细胞后其的免疫调节作用的变化。方法:实验于2006-06/2006-11在美国阿肯色大学医学院基因治疗中心完成。①实验方法:取健康人外周血(自愿捐献),采用改良Ficoll密度梯度离心的方法分离外周血单个核细胞,取贴壁细胞,分为rAAV/CEA转染组和空白对照组,均采用重组人粒细胞巨噬细胞集落刺激因子、白细胞介素4、肿瘤坏死因子-α诱导树突状细胞前体细胞成熟,将成熟树突状细胞与末梢血淋巴细胞按比例混合培养,可得到激活的细胞毒性T淋巴细胞。②实验评估:采用流式细胞仪检测树突状细胞表面标志表达,细胞毒性T淋巴细胞的免疫表型变化,细胞毒性T淋巴细胞γ-干扰素表达。采用酶联免疫吸附法(ELISA法)检测树突状细胞的白细胞介素12和白细胞介素10表达。结果:①rAAV/CEA转染树突状细胞的CD14较空白对照组降低,共刺激分子CD40、CD80、CD83、CD86表达均较空白对照组高。②成熟树突状细胞细胞因子表达中,rAAV/CEA转染组的白细胞介素12较空白对照组升高,白细胞介素10降低。③与空白对照组比较,rAAV/CEA转染组能高表达CD8+T细胞和其表型CD69,CD8/CD56的T细胞比例上调,CD25+CD4+的T细胞减少。④rAAV/CEA转染的细胞毒性T淋巴细胞表达相对较高水平的γ-干扰素,与杀伤实验结果相吻合。结论:rAAV/CEA转染树突状细胞能够激活细胞毒性T淋巴细胞的特异性杀伤作用,与树突状细胞和细胞毒性T淋巴细胞等免疫细胞表面标志变化和细胞内细胞因子水平变化相关。
Objective: In recent years, dendritic cells are involved in the regulation of more research. The aim of the experiment was to verify the effect of recombinant adeno-associated virus (rAAV) on the immunoregulation of dendritic cells transfected with antigens of carcinoembryonic antigen. METHODS: The experiment was performed at Gene Therapy Center, University of Arkansas Medical School, USA from June 2006 to November 2006. ①Experimental methods: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers by using modified Ficoll density gradient centrifugation. The cells were divided into rAAV / CEA transfection group and blank control group, Human granulocyte-macrophage colony-stimulating factor, interleukin-4 and tumor necrosis factor-α induce the maturation of dendritic cell precursors, and the mature dendritic cells and peripheral blood lymphocytes are mixed and cultured in proportion to obtain activated Cytotoxic T lymphocytes. ② Experimental evaluation: Flow cytometry was used to detect the expression of dendritic cell surface markers, the immunophenotypic changes of cytotoxic T lymphocytes and the expression of γ-interferon of cytotoxic T lymphocytes. The expression of interleukin-12 and interleukin-10 in dendritic cells was detected by enzyme linked immunosorbent assay (ELISA). Results: ① The CD14 of dendritic cells transfected with rAAV / CEA was lower than that of blank control group, and the expressions of costimulatory molecules CD40, CD80, CD83 and CD86 were higher than that of blank control group. ② In mature DCs, interleukin-12 in rAAV / CEA transfection group was higher than that in blank control group, and interleukin-10 was decreased. ③ Compared with the blank control group, the percentage of T cells that expressed CD8 + T cells and its phenotype CD69 and CD8 / CD56 was increased in rAAV / CEA transfected group, while T cells in CD25 + CD4 + cells decreased. ④AAV / CEA transfected cytotoxic T lymphocytes expressed a relatively high level of interferon-γ, consistent with the killing test results. CONCLUSION: Dendritic cells transfected with rAAV / CEA can activate cytotoxic T lymphocytes specifically, which is associated with changes of immune cell surface markers such as dendritic cells and cytotoxic T lymphocytes and changes of intracellular cytokine levels.