中文摘要：

目的：制备一种新型负载HBsAg基因的外切体（exosome）瘤苗,并探讨其生物学特性、免疫学功能。方法：运用分子克隆和病毒载体转染HBsAg基因构建AdVHBsAg-DC肝癌瘤苗,采用流式细胞术鉴定转染基因表达;提取exosome;以透射电镜观察、Western blot法鉴定exosome。结果：构建的重组AdVHBsAg腺病毒载体,经PCR和酶切鉴定,结果显示HBsAg基因片段已正确插入腺病毒载体中。包装的腺病毒载体具有良好的感染性,可以在293细胞中形成病毒颗粒。提取的exosome在透射电镜下可观察到直径为50-100nm的膜性微囊,圆形或椭圆形,有完整包膜。Western blot检测结果表明,exosome含有DC的特征分子和HBsAg基因。结论：成功制备并获得HBsAg基因修饰树突状细胞为来源的exosome瘤苗,为治疗乙肝相关肝癌提供新的思路。

英文摘要：

Objective：To obtain exosomes derived from adenovirus-mediated HBsAg gene-modified dendritic cells.Methods： Full length HBsAg cDNAs were cloned into shuttle2 vector.The HBsAg gene fragments resulted from the-S digested with PI-Sce and I-Ceu were linked to the linear adeno-X virus DNA.After packaged with HEK293 cells,the adenovirus expression vector was obtained.Then the recombinant adenovirus expression plasmid AdVHBsAg was transfected into human monocyte-derived dendritic cells.The exosomes were isolated from supematant of transfected DCs.Transmission electron microscopy was used to observe their structures.The expressions of several proteins were investigated by flow cytometry.Results： The shuttle2-S showed that band with 630 bp by digested with PI-Sce and I-Ceu,HBsAg gene in the inserted DNA of AdVHBsAg was confirmed by PCR,and predictive fragments proved by restriction enzyme digestion analysis were exhibited.CPE appear 10 after days HEK293 cells transfected AdVHBsAg.Application of the isolation procedure to transfected DCs revealed exosome vesicles by transmission electron microscopy.Protein analysis by Western blot was performed and revealed that the costimulatory molecule CD86,CD83 and HBsAg was detectable.Conclusion： The exosomes derived from HBsAg-DC may be a tool of the HBV related hepatocellular carcinoma immunotherapy.