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目的:观察荷载人生存素(survivin)的多表位树突状细胞(DC)疫苗的抗肿瘤活性。方法:分别将含4个sur-vivin的HLA-A2类限制性CD8+CTL表位和1个CD4+Th细胞表位的重组真核表达质粒pPIRESneo3.0-survivin(4)/Th,以及含4个CD8+CTL表位的重组质粒pPIRESneo3.0-survivin(4),转染人DC并制备DC疫苗。实验分为survivin(4)/Th组、survivin(4)组、空质粒组、未转染DC与T细胞共培养组和单独T淋巴细胞组。DC疫苗作用后,MCF-7细胞的凋亡率明显高于survivin(4)组(P<0.05),亦明显高于空质粒组、未转染DC与T细胞共培养组和单独T淋巴细胞组(P<0.05)。运用流式细胞术(FCM)分别检测DC表面CD83、CD86、T淋巴细胞表面CD4、CD8a的表达以及DC疫苗作用后MCF-7乳腺癌细胞的凋亡。用ELISA法检测上清中IFN-γ的含量。用四甲基偶氮唑蓝(MTT)比色法检测DC疫苗诱导的CTL对MCF-7细胞的抑制率。结果:流式细胞术检测显示,人DC高表达CD83、CD86;人外周血T淋巴细胞高表达CD4、CD8a;survivin(4)/Th组IFN-γ的含量[(66.50±3.34)ng/L]明显高于survivin(4)组[(46.10±1.35)ng/L]、空质粒组[(25.17±0.32)ng/L]、未转染DC与T细胞共培养组[(25.47±0.95)ng/L]和单独T淋巴细胞组[(23.73±0.50)ng/L],P<0.05。survivin(4)/Th组中MCF-7细胞的抑制率明显高于survivin(4)组、空质粒组、未转染DC与T细胞共培养组和单独T淋巴细胞组(P<0.05)。DC疫苗作用后,MCF-7细胞的凋亡率[(10.63±0.29)%]明显高于survivin(4)组(P<0.05),亦明显高于空质粒组、未转染DC与T细胞共培养组和单独T淋巴细胞组(P<0.05)。结论:荷载多个survivin的CD8+CTL表位的树突状细胞肿瘤疫苗具有很强的抗肿瘤活性,CD4+Th细胞对CD8+CTL的抗肿瘤方面有明显的促进作用。
Objective: To observe the antitumor activity of multi-epitope dendritic cell (DC) vaccine loaded with human survivin. Methods: The recombinant eukaryotic expression plasmid pPIRESneo3.0-survivin (4) / Th containing 4 sur-vivin HLA-A2 restricted CD8 + CTL epitopes and 1 CD4 + Th cell epitope, respectively, Four CD8 + CTL epitopes of the recombinant plasmid pPIRESneo3.0-survivin (4) were transfected into human DCs and DC vaccines were prepared. The experiment was divided into survivin (4) / Th group, survivin (4) group, empty plasmid group, non-transfected DC and T cell co-culture group and T lymphocyte alone group. The apoptosis rate of MCF-7 cells after DC vaccine treatment was significantly higher than that of survivin (4) group (P <0.05), also significantly higher than that of empty plasmid group, non-transfected DC and T cell co-culture group and T lymphocyte alone Group (P <0.05). The expression of CD4 and CD8a on the surface of CD83, CD86 and T lymphocytes on DCs and the apoptosis of MCF-7 breast cancer cells on DCs were detected by flow cytometry (FCM). The content of IFN-γin the supernatant was detected by ELISA. The inhibitory rate of DC vaccine-induced CTL on MCF-7 cells was detected by MTT assay. Results: The results of flow cytometry showed that human DCs highly expressed CD83 and CD86, and the levels of IFN-γ in peripheral blood T lymphocytes were significantly higher than that of CD4 and CD8a (66.50 ± 3.34 ng / L ] Was significantly higher than those in survivin group [(46.10 ± 1.35) ng / L] and [(25.17 ± 0.32) ng / L) ng / L] and T lymphocyte alone group [(23.73 ± 0.50) ng / L], P <0.05. The inhibitory rate of MCF-7 cells in survivin (4) / Th group was significantly higher than that in survivin (4) group, empty plasmid group, non-transfected DC and T cell co-culture group and T lymphocyte alone group (P <0.05). The apoptosis rate of MCF-7 cells was significantly higher than that of survivin (4) group (P <0.05), and also significantly higher than that of empty plasmid group and non-transfected DC and T cells Co-culture group and T lymphocyte alone group (P <0.05). Conclusion: The dendritic cell tumor vaccine carrying multiple survivin CD8 + CTL epitopes has strong antitumor activity, and CD4 + Th cells have a significant promotion on anti-tumor of CD8 + CTL.