间质干细胞exosome转运14-3-3ζ抗肝纤维化的作用及机制

There is still no ideal antifibrotic agent available and mesenchymal stem cells (MSCs) transplantation has recently gained increasing attention in liver fibrosis. We found that MSCs derived exosomes (MSC-exosome), an active component secreted by MSCs, could ameliorate carbon tetrachloride induced liver fibrosis in mice. However, the mechanisms for this amelioration remain unknown. Our preliminary study suggested that MSC-exosome exert antifibrotic effect through exosome mediated 14-3-3ζtransfer to myofibroblast (MFB). Previous studies have demonstrated that 14-3-3ζ can reduce YAP nuclear localization and YAP plays a key role in collagen synthesis of MFB. Based on these results and analysis of bioinformatics, we hypothesize that MSC-exosome derived 14-3-3ζ can suppress LOXL2 expression through inhibition of YAP nuclear localization, and thus decrease collagen synthesis and deposition in liver fibrosis. We will confirm the above hypothesis using experiments in vitro and in vivo to explore the role and mechanism of 14-3-3ζ from MSC-exosome in liver fibrosis. Then 14-3-3ζ overexpressed MSC-exosome will be produced and developed as a new strategy for treatment of liver fibrosis, which might provide evidence for MSC-exosome based antifibrotic therapy and a new therapeutic agent for liver fibrosis.

肝纤维化治疗目前尚缺乏理想的方法，间质干细胞（MSCs）在肝纤维化治疗中的作用越来越引起人们的重视。我们发现脐带MSCs分泌的外泌体（MSC-exosome）具有抗肝纤维化功能，然而作用机制并不清楚。预研究表明14-3-3ζ蛋白可能是MSC-exosome中抗肝纤维化的重要活性分子，且有研究发现14-3-3ζ可阻滞YAP入核，YAP核定位与肝纤维化胶原合成密切相关。基于前期研究和生物信息学分析，我们提出假说：在肝肌成纤维细胞中，MSC-exosome转运的14-3-3ζ通过抑制YAP入核下调LOXL2表达，降低胶原合成。本项目拟采用Ch-IP、Co-IP、双荧光素酶报告基因等技术揭示MSC-exosome转运14-3-3ζ抗肝纤维化的机制，并建立以MSC-exosome为载体过表达14-3-3ζ抗肝纤维化的方法，为MSC-exosome抗肝纤维化提供依据，为肝纤维化治疗提供新的策略。

我们发现脐带MSCs分泌的外泌体（MSC-exosome）具有抗肝纤维化功能，然而作用机制并不清楚。14-3-3ζ蛋白可能是MSC-exosome中抗肝纤维化的重要活性分子，且有研究发现14-3-3ζ可阻滞YAP入核，YAP核定位与肝纤维化胶原合成密切相关。本项目旨在探明 MSC-exosome 转运 14-3-3ζ抗肝纤维化的分子机制，为MSC-exosome 抗肝纤维化的生物疗法提供实验和理论依据。按照项目计划书规定的研究内容，本项目完成了以下研究：1）分离纯化获得MSC-exosome（MSC-ex）、14-3-3ζ-MSC-exosome（14-3-3ζ-ex），进行生物学功能评价；2）评价MSC-ex、14-3-3ζ-ex的抗肝纤维化疗效；3）明确了14-3-3ζ-ex阻滞YAP核定位抑制LOXL2表达的作用；4）阐明TGF-β诱导YAP 核定位, 转录调控LOXL2 表达的机制；5）探讨了YAP/LOXL2 在肝纤维化进展判断中的作用。同时，本项目进一步探讨了MSC-ex转运miR-373下调TGFR2表达，抑制TGF-β诱导的YAP活化及LOXL2表达的作用。结果表明：1）miR-373调控TGF-βR2抑制TGF-β/SMAD信号通路；2）MSC-ex调控小鼠肝组织miR-373，抑制TGF-β/YAP/LOXL2表达；3）miR-373修饰可增强MSC-ex的抗LOXL2作用。此外，本项目对MSC-ex在非酒精性脂肪肝、肝癌及淋巴水肿修复中的作用进行了拓展。结果表明：1）MSC-ex抑制肝纤维化向肝癌进展的作用；2）MSC-ex调控肝组织脂肪代谢减缓非酒精性脂肪肝的作用及机制；3）MSC-ex可转运Ang2促进淋巴管再生，修复淋巴水肿的作用及机制。对照项目计划书，我们已建立稳定高效的 14-3-3ζ-ex、miR373-ex抗肝纤维化的方法，为肝纤维化的治疗提供新的模式和研究策略；已初步探明MSC-ex 转运 14-3-3ζ、miR-373下调TGF-β诱导的YAP/LOXL2通路抗肝纤维化的分子机制，为MSC-ex抗肝纤维化的生物疗法提供实验和理论依据。