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目的:以GPC3基因转染人外周血单个核细胞来源的树突状细胞(DCs),诱导CTL产生特异性抗肝癌免疫。方法:从HLA-A2正常健康成人外周血分离单个核细胞,经GM-CSF和IL-4诱导产生DCs;通过脂质体转染法将携带人GPC3基因的质粒pEF-hG-PC3转染DCs,蛋白质印迹法检测磷脂酰肌醇蛋白聚糖-3表达,流式细胞仪检测DCs表型的变化;MTT法检测DC诱导CTL对人肝癌细胞HepG2的特异性杀伤。结果:经细胞因子诱导产生了具有典型形态学的DCs,经肿瘤坏死因子-α(TNF-α)刺激成熟后可高表达CD80、CD86及CD83,而转染GPC3基因对协同刺激因子在DCs中的表达的影响并不显著。转染GPC3基因后,DCs可促进CTL特异性杀伤HepG2细胞,在不同效靶比的杀伤率:100∶1为(38.90±0.95)%,50∶1为(30.83±1.24)%,10∶1为(20.84±0.65)%,明显高于空载体组和对照组,P<0.05。结论:GPC3基因转染人外周血单个核细胞来源的DCs可诱导和促进CTL特异性杀伤肝癌细胞HepG2作用,为治疗肝癌提供了新的免疫靶点。
OBJECTIVE: To transduce dendritic cells (DCs) derived from human peripheral blood mononuclear cells with GPC3 gene and induce CTL to produce specific anti-liver cancer immunity. Methods: Mononuclear cells were isolated from peripheral blood of healthy normal adult HLA-A2 and DCs were induced by GM-CSF and IL-4. Plasmids pEF-hG-PC3 carrying human GPC3 gene were transfected into DCs by lipofection , The expression of glypican-3 was detected by Western blotting, the phenotype of DCs was detected by flow cytometry, and the cytotoxicity of CTL on HepG2 cells was detected by MTT assay. RESULTS: DCs with typical morphology induced by cytokines were highly expressed in CD80, CD86 and CD83 after stimulated by tumor necrosis factor-α (TNF-α) The impact of expression is not significant. After transfection with GPC3 gene, DCs could promote CTL specific killing of HepG2 cells. The killing rates of CTLs at different target ratios were (38.90 ± 0.95)%, 50:1 (30.83 ± 1.24)%, 10:1 (20.84 ± 0.65)%, which was significantly higher than that of the empty vector group and the control group (P <0.05). Conclusion: GPC3 gene transfected human peripheral blood mononuclear cells derived DCs can induce CTL-specific Hepatoma cell HepG2 role in the treatment of liver cancer provides a new immunological target.