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目的:探讨腺病毒负载SOCS1siRNA和IL-12基因共同修饰人喉癌细胞(Hep-2)抗原致敏的树突状细胞(DC)在体外诱导﹑激活细胞毒性T淋巴细胞(CTL)免疫杀伤Hep-2的效能及相关免疫机制。方法:采用重组腺病毒介导SOCS1siRNA基因和重组腺病毒介导IL-12基因共同修饰人外周血单个核细胞(PBMC)来源的DC;采用反复冻融法提取Hep-2粗体抗原(Ag)致敏的基因修饰的DC;用ELISA法检测各组DC及CTL分泌IL-12和IFN-γ的水平;MTT法检测DC刺激同源T淋巴细胞增殖能力及CTL免疫杀伤Hep-2的效能;统计学分析各组间的差异。结果:DC体外诱导培养成功,Ad-GFP转染可见荧光表达率在90%以上。经SOCS1siRNA和IL-12基因共同修饰能有效下调DC中SOCS1蛋白的表达和上调IL-12蛋白的表达。IL-12因子的分泌率也明显高于SOCS1siRNA或IL-12的单基因转染,并能促使T细胞显著增殖和活化。DC和CTL持续高分泌IFN-γ,因此产生对Hep-2的特异性细胞免疫。结论:SOCS1siRNA和IL-12基因共同修饰喉癌抗原致敏的DC分泌的IL-12和IFN-γ的能力增强。双基因共同修饰喉癌抗原致敏的DC与T细胞混合反应能有效的促进T细胞增殖,促进其分泌IFN-γ和IL-12,从而诱导CTL更强的特异性杀伤喉癌细胞的能力。
Objective: To investigate the effect of dendritic cells (DCs) sensitized by adenovirus loaded SOCS1 siRNA and IL-12 gene on Hep-2 antigen-induced activation of cytotoxic T lymphocytes (CTLs) in vitro. Hep- -2 performance and related immune mechanisms. Methods: The DCs derived from human peripheral blood mononuclear cells (PBMCs) were co-modified by recombinant adenovirus mediated SOCS1siRNA gene and recombinant adenovirus mediated IL-12 gene. Hep-2 antigen (Ag) was extracted by repeated freeze- The level of IL-12 and IFN-γ secreted by DCs and CTLs in each group were detected by ELISA. The proliferation of DCs stimulated by DCs and the efficacy of CTLs killing Hep-2 were detected by MTT assay. Statistical analysis of differences between groups. Results: DCs were successfully induced in vitro. The expression of Ad-GFP reached over 90%. The SOCS1 siRNA and IL-12 gene co-modified can effectively down-regulate SOCS1 protein expression in DC and up-regulate IL-12 protein expression. The secretion rate of IL-12 was also significantly higher than that of SOCS1 siRNA or IL-12, and could promote the significant proliferation and activation of T cells. DCs and CTLs consistently secrete IFN-γ, thus producing specific cellular immunity to Hep-2. Conclusion: The ability of SOCS1 siRNA and IL-12 gene to co-modify IL-12 and IFN-γ secreted by laryngeal cancer antigen-primed DCs is enhanced. The double gene co-modified laryngeal cancer antigen-sensitized DC mixed with T cells can effectively promote the proliferation of T cells, and promote the secretion of IFN-γ and IL-12, thereby inducing CTL stronger and more specific killing laryngeal cancer cells.