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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(dendriticcell,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;Westernblot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DCCD1a、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Westernblot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。
Background and objective Currently, there is a lack of effective immunotherapy for hepatocellular carcinoma associated with hepatitis B virus infection. Tumor-specific immunotherapy based on dendritic cells (DC) provides a novel method for immunotherapy of hepatocellular carcinoma associated with hepatitis B virus infection. In this study, HBsAg-DC vaccine was prepared by transfecting DCs with recombinant plasmids carrying HBsAg in vitro and evaluated the cytotoxic T lymphocyte responses against HepG2.2.15 induced by HBsAg-DC in vitro. Methods The recombinant plasmid pCR3.1-S containing HBsAg gene was transfected into DCs on the fifth day. Western Blot and immunofluorescence were used to identify the transfected cells. The DC-induced anti-HepG2.2.15 specific cytotoxicity effect. Results The results of cell phenotype identification showed that the expression of DCCD1a, CD11c, CD86, CD80 and HLA-DR were 55.0%, 98.6%, 86.1%, 66.1% and 88.9% respectively on the 5th day after induction. The results of immunofluorescence and Western blot showed that HBsAg The results of specific cytotoxicity assay showed that different effective target ratios were detected by MTT assay and pCR3.1-S transfected DC group showed highly specific and specific killing activity on HepG2.2.15 hepatoma cells, killing rate 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% : 1), which was significantly higher than that of pCR3.1-DC group and DC group (P <0.05, n = 4). Conclusion The recombinant plasmid transfected DC can express HBsAg efficiently and induce specific CTL reaction in vitro.