HBsAg基因修饰的树突状细胞体外诱导抗 HepG2.2.15特异性细胞毒作用

背景与目的:目前缺少一种针对乙型肝炎病毒感染相关肝癌的有效免疫治疗手段.以树突状细胞( dendritic cell, DC)为基础的肿瘤特异性免疫治疗方法,为免疫治疗乙型肝炎病毒感染相关的肝癌提供了新方法 .本实验通过体外负载乙型肝炎表面抗原基因( HBsAg)的重组质粒转染 DC,制备 HBsAg-DC瘤苗,评价 HBsAg-DC瘤苗体外诱导抗 HepG2.2.15的细胞毒性 T淋巴细胞反应.方法:将已构建含 HBsAg基因的重组质粒 pCR3.1-S转染培养第 5天的 DC; Western blot和免疫荧光法鉴定转染基因表达;以 MTT法测定 DC诱导的抗 HepG2.2.15特异性细胞毒作用.结果:细胞表型鉴定结果显示诱导 5天的 DC CD1a、 CD11c、 CD86、 CD80和 HLA-DR表达量分别为 55.0%、 98.6%、 86.1%、 66.1% 和 88.9%;采用免疫荧光和 Western blot法研究表明 HBsAg基因能在转染的 DC中表达; MTT法检测特异性细胞毒作用结果显示:不同效靶比, pCR3.1-S转染 DC组均显示对 HepG2.2.15肝癌细胞高效特异的杀伤活性,杀伤率分别为:( 52.3± 2.8)%( E∶ T为 5∶ 1)、( 64.6± 2.4)%( 10∶ 1)、( 78.8± 2.6)%( 20∶ 1)、( 82.1± 2.4)%( 40∶ 1),显著高于 pCR3.1-DC 组和 DC组 (P< 0.05, n=4).结论:重组质粒转染的 DC能够有效表达 HBsAg;并在体外诱导出特异性 CTL反应.

Cytotoxicity induced by HBsAg gene modified-dendritic cells against hepatocellular carcinoma cell HepG2.2.15

BACKGROUND & OBJECTIVE: Up to now, there is no efficient immunotherapy for hepatocellular carcinoma (HCC). Dendritic cell (DC) vaccine could be a potential tool for HCC immunotherapy. This study was to evaluate the effect of dendritic cells (DCs) transfected with recombinant plasmid bearing hepatitis B virus surface antigen (HBsAg) gene, and the capability of generating specific cytotoxic T lymphocytes (CTL) response against HepG2.2.15 in vitro, which were induced by genetically modified DCs. METHODS: After cultured for 5 days, the DCs were transfected with pCR3.1-S by liposome. The HBsAg gene expression on pCR3.1-transfected DCs was identified by Western blot analysis, and immunofluorescence methods. The cytotoxicity against HepG2.2.15, which were induced by DCs, was tested by MTT assay. RESULTS: DCs up-regulated the expression of CD1a (55.0%), CD11c (98.6%), CD86 (86.1%), CD80 (66.1%), and HLA-DR (88.9%) after cultured for 5 days. Indirect immunofluorescence, and Western blot analysis showed that HBsAg gene was expressed on transfected DCs. The death rates of HepG2.2.15 cells induced by DCs transfected with pCR3.1-S were (52.3+/-2.8)% (E:T=5:1), (64.6+/-2.4)% (10:1), (78.8+/-2.6) (20:1), (82.1+/-2.4)% (40:1), while the pCR3.1- transfected and non-transfected DCs only induced relatively lower cytotoxicity (P< 0.05, n=4). CONCLUSION: DCs transfected with recombined plasmid expressed HBsAg efficiently, and the genetically modified DCs evoke a higher CTL response in vitro.