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目的:研究EPHA2基因修饰的树突状细胞(dendritic cell,DC)疫苗诱导细胞毒性T淋巴细胞(cytotoxic T lympho-cyte,CTL)对U251胶质瘤细胞的杀伤效应,为胶质瘤的免疫治疗提供新的方法。方法:将重组EPHA2腺病毒rAd-EPHA2感染HLA-A2阳性的人外周血来源的DC,制备EPHA2基因修饰的DC疫苗,Western blotting和FACS方法检测感染后DC的EPHA表达。以DC疫苗体外刺激HLA-A2阳性的单个核细胞,酶联免疫斑点实验(enzyme-linked immunospot assay,ELISPOT)和标准51Cr释放实验分别检测DC疫苗所诱导的CTL活性和对HLA-A2阳性的U251细胞的杀伤作用(另设rAd-Lac Z感染的DC组和PBS组作为对照)。结果:成功制备了EPHA2基因修饰的DC疫苗,其可有效表达EPHA2蛋白。与感染rAd-LacZ的DC和PBS组相比,感染rAd-EPHA2的DC疫苗能有效激发CTL活性[(187±21)vs(12±4)、(18±5)个,P<0.01];所诱导的CTL对胶质瘤U251细胞有明显的杀伤效应[(45.7±6.8)%vs(7,1±4.5)%,P<0.01],对自身淋巴细胞没有杀伤效应。结论:EPHA2基因修饰的DC疫苗能有效激发CTL活性,并对胶质瘤U251细胞有明显的杀伤活性。
OBJECTIVE: To study the killing effect of cytotoxic T lympho-cyte (CTL) induced by EPHA2 gene-dendritic cell (DC) vaccine on U251 glioma cells and to study the immunotherapy of glioma Provide new methods. Methods: Recombinant EPHA2 adenovirus rAd-EPHA2 was used to infect HLA-A2-positive DCs derived from human peripheral blood to prepare DC vaccine modified by EPHA2 gene. EPHA expression was detected by Western blotting and FACS. HLA-A2-positive mononuclear cells were stimulated with DC vaccine in vitro. ELISPOT and 51Cr release assay were used to detect the CTL activity induced by DC vaccine and the activity of HLA-A2 positive U251 The killing effect of cells (rAd-Lac Z infected DC group and PBS group as control). Results: EPHA2 gene modified DC vaccine was successfully prepared, which can effectively express EPHA2 protein. DC vaccines infected with rAd-EPHA2 could effectively stimulate CTL activity compared with DCs and rAd-LacZ infected groups ([187 ± 21] vs (12 ± 4), (18 ± 5), P <0.01] The induced cytotoxicity of CTL on glioma U251 cells was significantly (45.7 ± 6.8)% vs (7 ± 4.5)%, P <0.01]. No cytotoxic effect was observed on the autologous lymphocytes. Conclusion: EPHA2 gene modified DC vaccine can effectively stimulate CTL activity and have obvious cytotoxic activity on glioma U251 cells.