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目的探讨HBsAg重组腺病毒Ad-S转染的小鼠树突状细胞(DC)诱导HBV转基因(Tg)小鼠免疫应答的作用特点及其可能的治疗作用。方法Tg小鼠的骨髓细胞体外扩增为DC,转染Ad-S或被HBsAg蛋白冲击后,与pcDNA3．1(+)-S质粒分别免疫Tg鼠,用流式细胞术、乳酸脱氢酶释放法、酶联免疫分析(ELISA)、荧光定量聚合酶链反应(PCR)等分别检测脾脏T细胞内细胞因子和细胞毒性T淋巴细胞(CTL)活性、血清HBsAg、抗-HBs、HBV DNA及丙氨酸转氨酶(ALT)水平；病理和免疫组化分析肝脏组织学及HBsAg和HBcAg表达。结果免疫后1～2周,DC/Ad-S比DC/HBsAg和pcDNA3．1(+)-S诱导Tc分泌干扰索γ(IFN-γ)和特异性CTL均显著增强,而DC/ HBsAg组CTL较pcDNA3．1(+)-S组增强(P＜0．05)；DC/HBsAg免疫后1、2、4周CTL迅速减弱,DC/Ad-S在1、2周CTL差异不明显,但至4周时明显减弱。免疫后1～4周,对Tg鼠血清HBsAg、HBV DNA及肝组织HBcAg和HBsAg表达的抑制作用最强为DC/Ad-S免疫,其次为DC/HBsAg,显著强于pcDNA3．1(+)-s(P＜0．05或P＜0．01)。肝脏组织学、血清抗-HBs和ALT水平各组差异无统计学意义。结论Ad-S转染的DC比HBsAR冲击的DC和DNA疫苗诱导更强的Tcl和CTL应答,能较迅速抑制HBV转基因小鼠血清HBsAg、HBV DNA和肝脏HBcAg和HBsAg表达。
Objective To investigate the immunological response of HBV transgenic mice induced by dendritic cells (DCs) transfected with recombinant adenovirus Ad-S and its possible therapeutic effects. Methods Tg mouse bone marrow cells were expanded to DC in vitro. After being transfected with Ad-S or HBsAg protein, Tg mice were immunized with pcDNA3.1 (+) - S plasmid respectively. The expression of lactate dehydrogenase Release assay, enzyme-linked immunosorbent assay (ELISA) and fluorescence quantitative polymerase chain reaction (PCR) were used to detect the cytokines and cytotoxic T lymphocytes (CTL) activity in splenic T cells, serum HBsAg, anti-HBs, Alanine aminotransferase (ALT) levels; histopathological and immunohistochemical analysis of liver histology and HBsAg and HBcAg expression. Results The levels of IFN-γ and CTL induced by DC / Ad-S compared with DC / HBsAg and pcDNA3.1 (+) -S were significantly increased at 1 and 2 weeks after immunization, while DC / HBsAg group (P <0.05). The CTL of DC / HBsAg was weakened rapidly at 1, 2 and 4 weeks after immunization. The difference of CTL between DC and Ad-S at 1 and 2 weeks was not obvious, But significantly weakened by 4 weeks. The highest inhibitory effect on the expression of HBsAg, HBV DNA and HBcAg and HBsAg in the serum of DCs was DC / Ad-S immunization, followed by DC / HBsAg at 1 to 4 weeks after immunization, which was significantly stronger than that of pcDNA3.1 (+) -s (P <0.05 or P <0.01). Liver histology, serum anti-HBs and ALT levels in each group showed no significant difference. CONCLUSION Ad-S-transfected DCs induce stronger Tcl and CTL responses than DCs and DNA vaccines impacted by HBsAR, which can rapidly inhibit the expression of HBsAg, HBV DNA and HBcAg and HBsAg in liver of HBV transgenic mice.