强制泛素化乙肝病毒核心蛋白负载DC诱导HBV特异性CTL的机制研究

Chronic hepatitis B virus (HBV) infection is related to weak specific cellular immune response of host to HBV. Our previous studies have shown that when hepatitis B virus core protein (HBcAg) is fused to ubiquitin, its degradation by the ubiquitin-proteasome system (UPS) and presentation can be accelerated, and specific cellular immune responses to HBV can be induced. Our previous studies have also shown that cell penetrating peptides (CPPs) can effectively carry the antigen of HBV into dendritic cells (DCs), and upregulate HBV-specific CTL activity. On the basis of preliminary studies, We intend to immunize HBV transgenic mice with DCs loaded with purified fusion protein containing CPPs and ubiquitinated HBcAg or lentiviral vector encoding ubiquitinated HBcAg gene, in order to make HBcAg be internalized and rapidly degradated by UPS to be the epitope peptide suitable for presenting by major histocompatibility complex class I antigen presenting pathway, then, HBV-specific CTL responses of the body can be effectively stimulated. The aim of this study is to investigate the mechanism and capacity of DCs induced with ubiquitinated HBcAg mediated by CPPs or lentiviral vector to stimulate lymphocytes proliferation and generate stronger HBV-specific CTL responses. The results may be helpful in seeking novel approaches to the control of persistent HBV infection, and provide a new idea and experimental basis of immune therapy for chronic HBV infection.

乙肝病毒（HBV）感染的慢性化与机体特异性细胞毒T淋巴细胞（CTL）免疫应答低下有关。我们的前期研究显示，乙肝病毒核心蛋白（HBcAg）的强制泛素化可加速其在细胞内的降解并诱导特异性免疫反应，而细胞穿透肽可以快速、高效转导乙肝病毒相关抗原进入树突状细胞（DC），上调HBV特异性CTL活性。本研究拟在前期研究的基础上，将HBcAg强制泛素化，分别以细胞穿透肽和慢病毒为载体，体外负载DC，并免疫HBV转基因小鼠，旨在使HBcAg胞内化，并迅速被泛素-蛋白酶体系统（UPS）降解为最适于提呈的表位肽，经MHC-I类抗原提呈途径提呈，更有效地刺激机体产生HBV特异CTL反应。本课题将阐明强制泛素化HBcAg在UPS作用下诱导HBV特异性CTL反应的效果及其机制，为慢性乙型肝炎的抗病毒治疗提供新的策略和实验基础。

乙型肝炎病毒（HBV）感染是严重的公共卫生问题，但是，目前临床尚无满意的抗HBV药物，HBV感染的慢性化与机体特异性细胞毒T淋巴细胞（CTL）免疫应答低下有关。探索新的诱导HBV特异性细胞免疫的方法有广阔的临床应用前景。本课题构建了表达细胞穿透肽(CTP)与泛素化HBcAg的融合蛋白（Ub-HBcAg-CTP）以及包装了负载泛素化HBcAg的重组慢病毒（LV-Ub-HBcAg）。 Ub-HBcAg-CTP融合蛋白致敏的DC能有效刺激T细胞内细胞因子IFN-γ和IL-2的分泌水平，可以明显提高淋巴细胞中Thl型细胞的数量并能有效增加细胞毒T淋巴细胞的表达；融合蛋白Ub-HBcAg-CTP免疫BALB/c小鼠后，不仅能有效刺激小鼠体内T淋巴细胞的增殖能力，而且可以明显提高T淋巴细胞分泌Thl型细胞因子的水平，并能有效增加病毒特异性CTL的表达及活性，具体机制可能与JAK/STAT信号通路活化有关；融合蛋白Ub-HBcAg-CTP免疫HBV转基因小鼠后可诱导HBV特异性CTL抑制HBV复制。重组慢病毒LV-Ub-HBcAg体外修饰的DC免疫和直接免疫两种免疫方式免疫BALB/c小鼠后，均可诱导以IgG2α为主的抗-HBc抗体反应，刺激Th1型细胞因子的分泌，促进T淋巴细胞增殖，增强特异性CTL活性。重组慢病毒LV-Ub-HBcAg免疫HBV转基因小鼠后可诱导以IgG2α为主的HBcAb水平，刺激Th1型细胞因子的分泌，诱导Th1型免疫反应，增强特异性CTL活性，抑制HBV复制和抗病毒作用，LV-Ub-HBcAg上调T-bet的表达和下调GATA-3的表达可能参与Th1分化。本研究将泛素与HBcAg融合使HBcAg强制泛素化，利用细胞穿透肽和慢病毒两种不同的介导方式，通过小鼠体外及体内实验，证实了强制泛素化HBcAg在泛素-蛋白酶体系统作用下诱导HBV特异性CTL反应的效果及其作用机制，为慢性乙型肝炎的抗病毒治疗提供新的策略和实验基础。