β3整合素、miR-122联合修饰肝星状细胞源性exosome以重塑"细胞间通讯网络"对肝纤维化的干预研究

De-differentiation of hepatic stellate cell (HSCs) and differentiation blockade of liver stem cells reflect the critical pathophysiological mechanisms of liver fibrosis. Exosome, which is secreted by HSCs, has recently been proved to responsible for these pathophysiological disorders by the 'communication network' between HSCs and liver stem cells. We, therefore, attempt to modify the HSC-derived exosomes by both β3 integrin and miR-122. Specific binding of β3 integrin and stem cell marker, CD90.1, results in the targeting communication between HSCs and liver stem cells. Transcellular transportation of miR-122, mainly from HSCs to liver stem cells, is then fulfilled by the fusion of cell membrane and exosome. Reconstruction of 'intercellular communication network' restores the miR-122 level in both HSCs and liver stem cells. It is expected to abolish the de-differentiation phenotype of HSCs, and also induce the differentiation from liver stem cells to hepatocytes. Liver fibrosis may be resultantly inhibited.

肝星状细胞（hepatic stellate cell, HSC）失去分化表型，以及肝干细胞向肝细胞分化受阻是诱导肝纤维化的关键病理生理机制。近期发现，细胞主动分泌形成的exosome介导了HSC-肝干细胞"细胞间通讯网络"，从而构成两者发生的共同基础。为此，本研究拟采用β3整合素、miR-122联合修饰HSC来源性exosome。借助β3整合素与肝干细胞标志物CD90.1特异性结合，实现HSC-肝干细胞的"靶向细胞间通讯"；并以靶向肝干细胞的exosome为载体，持续进行miR-122的跨细胞转运。由此在重塑"细胞间通讯网络" 的基础上纠正miR-122表达缺失，既恢复HSC的分化表型，又诱导肝干细胞向成熟肝细胞分化。最终通过多途径同步干预，发挥防治肝纤维化的作用。

肝星状细胞（hepatic stellate cell, HSC）失去分化表型，以及肝干细胞向成熟肝细胞分化受阻是诱导肝纤维化的重要病理生理机制。近年研究证实，细胞主动分泌形成的exosome可能介导了HSC-肝干细胞“细胞间通讯网络”，从而构成两者发生的共同基础。本项目通过HSC靶向性慢病毒感染及GFP标记，证实活化HSC（MFB）来源性exosome能够与肝干细胞融合，从而介导肝脏的HSC-肝干细胞间通讯。这一细胞间通讯机制参与了肝纤维化过程中HSC及肝干细胞的表型调控。为此，本项目构建携带α-SMA启动子的pre-miR-122重组慢病毒，通过在MFB中特异性表达成熟miR-122，抑制其活化（间质）表型和成纤维能力。同时分离、纯化包含成熟miR-122的MFB来源性exosome，并与肝干细胞共孵育，借此实现了miR-122由MFB向肝干细胞的跨细胞转运。针对干细胞标志物和肝细胞标志物的检测表明，基于HSC-肝干细胞间通讯的miR-122转运具有促进肝干细胞向肝细胞表型分化的作用。在此基础上采用肝干细胞标志物的特异性配体（β3整合素）修饰MFB，还能够增强HSC-肝干细胞之间的定向通讯能力。本项目进而以CCl4诱导肝纤维化模型动物，并采用β3整合素、α-SMA启动子驱动的pre-miR-122联合修饰MFB的策略进行体内干预。结果显示，利用HSC、肝干细胞的“靶向通讯”可对两者表型进行同步调控。组织病理学评价进一步证实，上述干预策略可发挥拮抗肝纤维化进展，改善肝脏组织学病变的目的。由此可见，重塑“细胞间通讯网络”以纠正miR-122表达缺失，既有助于恢复HSC的分化表型，又诱导肝干细胞向成熟肝细胞分化，可望成为防治肝纤维化的有效手段。