CD100-Plexin B信号通路促进病毒性肝炎肝纤维化的作用机制

Viral-hepatitis-induced liver fibrosis accompanied with extracellular fibrin deposition and liver nodular regeneration. Inflammation-specific hepatocyte signals plays an important role in this abnormal wound healing process. The role of CD100 in tissue repair has been discovered currently. Our experiment has confirmed that CD100-Plexin B signal was activated in inflammatory hepatocytes, which may influences hepatocyte proliferation, cellular localization and induces abnormal tissue regeneration. The activation of CD100-PlexinB signal would cause TGF-β1 / DAN expression change in hepatocyte, and affect the activation of hepatic stellate cells. For further confirm our hypothesis, we planed our experiment as: collecting blood and liver tessues from patients with viral hepatitis and liver fibrosis, analysis the fibrosis related cytokines; detecting the functions of CD100-Plexin B in hepatocyte relocalization of liver tissue; constructing CD100 and Plexin B gene-interference system and analysis the mechanism of CD100-PlexinB in fibrosis regulation; constructing mouse model to confirm the roles of CD100-Plexin B involved in hepatocyte relocalization, stellate cell activation and liver fibrosis. We hope the expected results could provide experimental evidences for studies of viral hepatitis induced liver fibrosis, as well as benefit the drug development for anti-fibrosis therapy.

病毒性肝炎肝纤维化是HBV/HCV等诱导的肝损伤修复异常。最新研究发现，CD100-PlexinB在损伤修复中发挥重要作用。本实验初步研究证实，肝脏炎症诱导CD100、PlexinB表达水平增加，激活肝细胞CD100-PlexinB信号转导。该信号的活化一方面可能通过干扰再生细胞的有丝分裂定位引起肝窦修复异常，另一方面可影响TGF-β1/DAN旁分泌诱导肝星状细胞活化，最终促进肝脏结节状再生和纤维化沉积等病理过程。依此提出本实验思路：在病毒介导的肝纤维化患者中检测CD100及相关因子的表达，分析CD100-PlexinB在肝细胞增殖定位、纤维化因子旁分泌过程中发挥的作用；构建CD100/PlexinB表达/干扰体系，在细胞及小鼠模型中探讨该信号诱导肝脏纤维沉积和结节状再生的分子机制；在小鼠模型中评价CD100信号阻断对肝纤维化的治疗作用及应用前景，为肝纤维化机制研究及药物研发提供实验依据。

病毒性肝炎肝纤维化是HBV/HCV等诱导的肝损伤修复异常。最新研究发现，CD100-PlexinB在损伤修复中发挥重要作用。课题前期初步证实：肝脏炎症诱导CD100、PlexinB表达水平增加，激活肝细胞CD100-PlexinB信号转导。该信号的活化一方面可能通过干扰再生细胞的有丝分裂定位引起肝窦修复异常，另一方面可影响TGF-β1/DAN旁分泌诱导肝星状细胞活化，最终促进肝脏结节状再生和纤维化沉积等病理过程。.依此假设，课题进行了针对以下内容研究：在病毒介导的肝纤维化患者中检测CD100及相关因子的表达，分析CD100-PlexinB在肝细胞增殖定位、纤维化因子旁分泌过程中发挥的作用；构建CD100/PlexinB表达/干扰体系，在细胞模型中探讨该信号诱导肝脏纤维沉积的分子机制，评价CD100信号阻断对肝纤维化的治疗作用。.课题的具体研究方法包括：（1）收集了HBV患者和健康者血清及PBMCs样本近135例，通过多因子ELISA检测技术和流式细胞仪检测技术，检测了包括CD100在内的12种细胞因子水平变化情况；检测了T细胞、B细胞、NK细胞、单核巨噬细胞、DCs及血小板上CD100和CD72等特征分子的表达情况，并在不同患者间进行了比较；（2）收集了人肝组织样本共计15例，通过western blot、RT-PCR、免疫荧光染色等技术对组织中的CD100及活化信号进行了分析；（3）在体外建立了细胞共培养膜型，建立了小鼠的CCl4肝纤维化膜型，在体外和体内对CD100在肝纤维化中的作用进行了分析和讨论。.课题得出的主要研究结果包括：（1）在HBV患者血清中，CD100水平显著降低，且与ALT/AST、TPO水平正相关；（2）在HBV患者PBMCs中，T/B细胞上CD100水平升高，APCs上表达降低，且B细胞上CD100与肝纤维化相关；（3）血小板CD100与肝纤维化相关；（4）CD100可通过PlexinB诱导肝细胞表达DAN蛋白，从而抑制肝星状细胞活化和肝纤维化，调控信号为ERK signal。.课题结论为：CD100可通过对肝细胞DAN蛋白的促进作用抑制肝纤维化的发生，这种机制可能为肝纤维化药物的研发提供新的实验依据和思路。