树突状细胞来源的exosome胞内转运泛素化修饰的丁肝病毒抗原诱导T细胞分化在抑制HDV复制中的作用及免疫机制研究

Recent studies demonstrated that the main reason of chronic viral infection was the virus could not been cleaned completely by weak specific cytotoxic T lymphocytes (CTL) responses in vivo. Effective induction of specific CTLs production can inhibit HDV replication. Dendritic cell derived exosomes (Dex) have the dual functions of immune regulation and protein transport and are used in the research of anti-tumor and viral infection. Our previous work has confirmed viral antigen peptide was fused to ubiquitin and could induce HBV specific CTL immune response effectively and inhibit viral replication. However, the specific immune mechanism is unclear. In this study, S-HDAg fused with ubiquitin is expressed on the surface of Dex by means of lysosomal associated membrane protein 2b (Lamp2b), and then S-HDAg is transported into the antigen presenting cells by the transmembrane function of exosome and is combined with MHC-I molecules after the degradation of UPS. Specific CTL immune response is induced to inhibit HDV replication in mice. Meanwhile, we will detect the expression level of downstream transcription factors by blocking the JAK/STAT pathway and explore the immune mechanism of CTLs production by JAK/STAT pathway. The results may be helpful in providing a new idea and experimental basis of immune therapy for HDV infection in the future.

大量研究证实体内特异性CTL反应低下导致病毒清除不彻底可能是病毒感染慢性化的主要原因，有效诱导特异性CTLs生成能抑制HDV复制。树突状细胞来源的外泌体(Dex)具有免疫调节及蛋白转运的双重功能，已被应用于抗肿瘤及病毒感染的研究。本课题组前期研究发现病毒抗原肽经强制泛素化修饰后，能有效诱导HBV特异性CTL免疫应答并抑制病毒复制，但具体免疫机制尚不清楚。本课题拟借助溶酶体相关膜蛋白Lamp2b的定位功能将强制泛素化修饰的丁肝病毒抗原(S-HDAg)表达于Dex表面，旨在通过外泌体负载S-HDAg跨膜导入抗原提呈细胞内，S-HDAg经UPS降解作用后与MHC-I类分子结合，诱导特异性CTL免疫反应抑制小鼠体内HDV复制，并通过阻断JAK/STAT通路对下游转录因子的表达水平进行检测，探讨JAK/STAT通路参与CTLs生成的免疫机制，为今后HDV感染的免疫治疗提供新的思路及实验基础。

目的：通过将Ub-S-HDAg-Lamp2重组慢病毒质粒转染树突状细胞（DCs），明确能否获得携带有S-HDAg的树突状细胞来源的外泌体（Ub-S-HDAg-Dexs）；探讨Ub-S-HDAg-Dexs能否诱导T淋巴细胞活化，增强适应性免疫反应，在HDV复制小鼠中具有抗病毒作用；探讨JAK/STAT信号通路参与S-HDAg-Dexs增强特异性CTL反应的作用机制。.方法：1.构建重组质粒H10833：pLenti-EF1a-EGFP-P2A-Puro-CMV-SP-Ub-S-HDAg-Lamp2-3Flag，进行慢病毒的包装、滴定、转染髓源性DCs，提取DCs上清液中外泌体并鉴定；2.将HDV Ⅰ型表达质粒经尾静脉注射入Tg-HBV 转基因小鼠，测定血清中HDV RNA表达量及肝脏损伤情况；3.将HDV感染小鼠分为6组，分别以Control-Dexs、Blank-Dexs、S-HDAg -Dexs、S-HDAg-Dexs+AG490，PBS，IFN-α进行治疗，治疗结束后，收集血清、肝脏和脾脏；检测各组血清中HDV RNA、HBsAg、ALT、AST、IL-12水平、组织病理、特异性CTL反应、JAK/STAT信号通路相关蛋白的表达，探讨JAK/STAT信号通路参与S-HDAg-Dexs增强特异性CTL反应的潜在机制。.结果：1.核酸测序证明重组质粒H10833构建成功；GFP表达率为70%-80%；流式细胞仪检测DCs表面分子标记物：CD11c为90.01%, CD80为75.35%, MHC-II为82.63%和CD86为93.57%，光学显微镜及投射显微镜观察到DCs表面具有星状多形性或树枝状突起；提取成熟DCs上清液中的外泌体后，透射电镜观察提取物中物质成囊泡状，NTA分析提取物的直径集中在104.7纳米，Western blotting显示提取物表达阳性外泌体标志物Alix、Hsp90,TG101及表达目的蛋白HDAg和Flag，不表达外泌体阴性标记Calnexin，通过荧光显微镜观察到DC2.4吞噬PKH26标记的外泌体随时间而逐渐增加，免疫荧光检测到DC2.4中含有Flag蛋白。2. 高压水动力法向M-Tg HBV转基因小鼠注射含编码HDV Ⅰ型基因组的质粒7天后，小鼠血清中的HDV RNA含量达106拷贝/ml;约30%肝细胞中表达HDAg。