HBV^+自噬小体疫苗治疗HBV急性感染的实验研究  

Study on therapeutic efficacy of autophagosome derived from HepG2.2.15 cells against acute HBV infection

作　　者：王璐[1] 薛萌[1] 殷鹏飞[1] 樊飞[1] 曹萌[1] 王立新[1] 

机构地区：[1]东南大学医学院病原生物学与免疫学系,江苏南京210009

出　　处：《东南大学学报（医学版）》2016年第5期647-653,共7页Journal of Southeast University(Medical Science Edition)

基　　金：国家自然科学基金资助项目(31370895;31170857;30972783)

摘　　要：目的:探讨转染HBV基因组的Hep G2.2.15细胞自噬小体(HBV+DRibbles)诱导HBV特异性免疫应答及其对HBV急性感染的治疗作用。方法:ELISA法检测HBV+DRibbles体外再刺激HBs Ag特异性效应细胞产生IFN-γ的水平;对HBV急性感染模型小鼠实施HBV+DRibbles、HBV+DRibbles联合DC免疫,ELISA法检测HBV抗原及抗原肽刺激免疫小鼠淋巴细胞产生IFN-γ的含量;ELISA法、荧光定量PCR法和酶法分别检测小鼠血清HBe Ag、HBV DNA、ALT和AST的水平;免疫组织化学法检测小鼠肝组织HBc Ag表达及免疫病理损伤。结果:与培养液对照组和HBs Ag蛋白刺激组相比,HBV+DRibbles刺激HBs Ag特异性效应细胞能够产生更高水平的IFN-γ(P=0.004);与未负载抗原的DC细胞对照组和负载HBs Ag的DC刺激组相比,DC负载HBV+DRibbles再刺激HBs Ag特异性CD4+、CD8+T细胞均能产生更高水平的IFN-γ(P<0.001)。与PBS对照组相比,HBV+DRibbles疫苗组和HBV+DRibbles联合DC免疫组均能诱导HBV急性感染模型小鼠产生HBV特异性免疫应答,明显降低血清HBe Ag、HBV DNA水平,减低HBc Ag+肝细胞比例,而ALT及AST水平未见明显差异,肝组织结构基本正常;但HBV+DRibbles疫苗组与HBV+DRibbles联合DC免疫组之间差异无统计学意义。结论:HBV+DRibbles作为HBV抗原载体,能够有效诱导DC对HBV抗原的交叉递呈;HBV+DRibbles诱导的HBV特异性细胞免疫应答对HBV急性感染具有一定的治疗作用。Objective： To investigate the ability of HBV antigens inducing DC cross-presentation and to elicit HBV specific immune response by Hep G2. 2. 15 cells（ HBV＋DRibbles） in vitro and its effectiveness in acute HBVinfected mouse model. Methods： HBs Ag-specific effector cells were re-stimulated with HBV＋DRibbles or soluble HBs Ag,then isolated CD4＋or CD8＋T cells were co-cultured with HBV＋DRibbles or HBs Ag preloaded DC. The levels of IFN- γ in supernatants were measured by ELISA. Lymphocytes from HBV＋DRibbles alone or in combination with DC immunized acute HBV-infected mice or unvaccinated control mice were re-stimulated with HBV antigens and peptides,and the amounts of IFN-γ in supernatant were analyzed by ELISA. The levels of serum HBe Ag,HBV DNA,ALT and AST were detected by ELISA,real-time PCR and enzyme method,respectively.The expression of HBc Ag in hepatocytes was determined by immunohistochemical staining and the percentage of HBc Ag＋hepatocytes was counted under microscope. The liver sections were stained with hematoxylin- eosin.Results： The HBV＋DRibbles stimulated HBs Ag specific lymphocytes produced much higher IFN-γ than soluble HBs Ag（ P = 0. 004）. Also,the CD4＋or CD8＋T cells re-stimulated by HBV＋DRibbles preloaded dendritic cells generated significant higher IFN-γ（ P 0. 001）. Levels of IFN-γ in supernatants of HBV antigens and peptides restimulated lymphocytes from the mice immunized with HBV＋DRibbles alone or combined with DC were much higher than that of control group. While the differences were not significant between the mice vaccinated with HBV＋DRibbles alone and in combination with DC. Compared with unvaccinated control group,both of the therapies could remarkably reduce the levels of HBe Ag and HBV DNA and decrease HBc Ag expression in hepatocytes and the percentage of HBc Ag＋hepatocytes,whereas the serum ALT and AST levels were not affected by vaccinations. The livers showed normal architecture and a mild inflammatory responses. No significant diff

关 键 词：乙型肝炎 乙型肝炎病毒 自噬小体 治疗性疫苗 小鼠 

分 类 号：R392.11[医药卫生—免疫学] R512.62[医药卫生—基础医学]