活体荧光成像监测载抗原树突状细胞疫苗体内抗HBV作用研究

The lack of suitable animal models severely hinders the study of the immune mechanism of HBV-casued infection and the evaluation of the efficiency of anti-virus-therapy. To overcome the obstacles described above, in this study,we try to establish a liver-specific fluc-HBV long-term expression animal model by the construction of an expression plasmid with fluc gene and HBV genome, based on the hydrodynamic gene delivery technology and the PhageΨ31 integrase system. When HBV-loaded DCs, also labelled by Gaussia, injected into the model, we can study the destribution, migration, homeing, and the location of injected DCs by gaussia imaging, at the same time, the clearance of HBV-expressing cells can also be monitered by the attenuation of fluc ROI. Based on this dual-imaging model, we can focus our research on the the specific killing mechanisms of immune effector cells versus target cells. The cuurent project will take the most advantages of the dual-imaging technology for the applications in tracking virus genome and immune cells in-vivo, which paves the way for the study of the interactions of DCs vaccines or T cells with HBV and can also be a basic plat for the evaluation of the efficiency of immune cell therapy.

缺乏合适的模式动物研究体系，严重阻碍了HBV免疫致病机制的研究，以及抗病毒免疫治疗效果的评价。对此本课题拟构建含有萤火虫荧光素酶（Fluc）的HBV基因组表达载体，基于水动力转染及噬菌体整合酶体系，建立Fluc-HBV基因组肝脏长期稳定表达小鼠模型。进而以膜锚定Gaussia荧光素酶标记载HBV抗原树突状细胞，将其输注入Fluc-HBV模型小鼠体内，通过小动物分子双荧光成像，既可实时监测载抗原树突状细胞在体内的分布、迁移、归巢、定位，又可实时监测树突状细胞激活的免疫效应细胞对HBV表达细胞的杀伤作用，在此基础上开展部分免疫效应细胞体内杀伤靶细胞的作用机制研究。本课题拟充分发挥活体双荧光成像技术在病毒基因组和免疫细胞体内示踪方面的特殊优势，将为DCs疫苗、T细胞与HBV体内相互作用研究、以及为免疫细胞体内治疗效果评价提供基础平台。

缺乏合适的模式动物研究体系，严重阻碍了HBV免疫致病机制的研究，以及抗病毒免疫治疗效果的评价。依托本项目建立了报告基因标记的乙型肝炎病毒体内长期稳定表达小鼠模型，并利用活体生物发光成像技术建立了树突状疫苗评价体系,并监测了监测DCs过继性输注后在体内的分布、迁移、转归等。采用不同粒径、形貌的功能化纳米金作为促进剂考察了其对树突状细胞的成熟及抗原递呈能力的影响，发现纳米金鸡尾酒促进的DCs主要向肝胰淋巴结归巢，并提升了DCs抗嗜肝病毒效果；进一步发现过继性输注携带乙肝抗原的DCs具有更强的抗乙肝病毒效果。总体来说，通过系统研究，建立和完善用于乙型病毒性肝炎防治的免疫治疗药物药效评价及机制研究的规范化动物模型体系，依托单位与珀金埃尔默仪器（上海）有限公司合作共建的小动物活体影像技术服务与研发中心为企事单位进行乙型病毒性肝炎相关药物研发提供技术支撑和服务。