PLGA超声微泡携rAAV介导siRNA调控巨噬细胞极化抗血吸虫肝纤维化的实验研究

Liver granuloma and fibrosis are the main pathological characteristic of schistosomiasis. Currently, there is no ideal drugs for liver fibrosis in schistosomiasis. Researches show that M2 polarized macrophages play an important role in the formation liver fibrosis induced by schistosome in mouse, and Notch signaling pathway is the key regulator of macrophages M1 versus M2 polarization. The preliminary experiments carried out by the applicant showed that the Jagged1/Notch1 signaling pathway was highly activated in macrophages within the liver granuloma induced by schistosome. We supposed that it had antifibrotic role of regulating macrophages polarization by inhibiting Jagged1/Notch1 signaling in liver fibrosis induced by schistosome. The applicant try to prepare PEGylated recombinant adeno-associated virus mediated siRNA targeting Jagged1 (rAAV-siJagged1) for encapsulation within polylactic/poly glycolic acid (PLGA) ultrasound microbubble, and transfect the PLGA microbubble into the M2 polarized macrophages or the liver of mice with liver fibrosis induced by schistosome using ultrasound targeted microbubble destruction (UTMD). The aim is to investigate the antifibrotic effect of inhibiting Jagged1/Notch1 signaling in liver fibrosis induced by schistosome, which may provide new thoughts for the research on pathogenesis and treatment of hepatic fibrosis in schistosomiasis.

血吸虫虫卵肉芽肿及继发肝纤维化是血吸虫病的主要病理特征，目前尚无理想的抗血吸虫肝纤维化的药物。研究表明M2极化的巨噬细胞在小鼠血吸虫肝纤维化形成中起重要作用，而Notch信号是调控巨噬细胞M1/M2极化的关键信号通路。申请者预实验发现：血吸虫虫卵肉芽肿内的巨噬细胞中Jagged1/Notch1信号活性显著上调，由此我们推测通过阻断Notch信号转导来调控巨噬细胞极化方向可能有抗血吸虫肝纤维化的作用。本课题拟构建干扰Jagged1的siRNA重组腺相关病毒载体（rAAV-siJagged1），经聚乙二醇修饰后包载于聚乳酸-羟基乙酸（PLGA）微泡内，运用超声靶向微泡击破（UTMD）介导PLGA微泡分别转染M2极化的巨噬细胞和靶向导入血吸虫肝纤维化小鼠肝脏，研究抑制Jagged1/Notch1信号活化对血吸虫肝纤维化的影响，为探究血吸虫肝纤维化的发病机制和靶向治疗提供新的思路。

已有研究表明M2极化的巨噬细胞在小鼠血吸虫肝纤维化形成中发挥重要作用，而Notch信号是调控巨噬细胞M1/M2极化的关键信号通路。本研究发现，在血吸虫肝纤维化小鼠肝组织中，肝内巨噬细胞高表达Jagged1，M2极化关键分子Arg-1的表达也显著增强。体外实验发现，血吸虫可溶性虫卵抗原（SEA）可诱导巨噬细胞株RAW264.7高表达Jagged1，Notch1和Hes1，从而激活Notch信号通路；同时，RAW264.7细胞表达IL-10和CD206上升，向M2极化。进一步研究发现，当用γ-分泌酶抑制剂（DAPT）特异性抑制Notch1/Jagged1信号的转导后，经SEA刺激的RAW264.7细胞分泌IL-10和CD206明显下降，向M1极化。本项目首次从细胞水平和动物模型两个层面，阐明Jagged1/Notch1信号通路对肝内巨噬细胞极化方向的调控作用及其在小鼠血吸虫肝纤维化中的作用机制。本研究仍在继续进行以超声微泡为载体介导RNAi靶向干预Jagged1/Notch1信号治疗小鼠血吸虫肝纤维化的体内实验，为将来可能的临床应用提供实验依据。