Let-7b-5p介导的自噬流活化在间充质干细胞外泌体调控慢加急性肝衰竭中肝细胞焦亡的作用机制研究

我们前期工作证实，间充质干细胞（MSC）治疗可提高慢加急性肝衰竭（ACLF）患者的存活率(Hepatology,2017)，但具体机制尚不明确。预实验结果显示：MSC及其外泌体组分可明显抑制ACLF小鼠肝细胞焦亡并诱导自噬流活化，而阻断自噬流活化后焦亡细胞显著增加。抑制MSC外泌体分泌导致自噬流活化受阻，细胞焦亡增加，提示MSC外泌体介导的自噬流活化可能在其治疗ACLF中扮演了重要角色。进一步对外泌体中microRNA测序发现Let-7b-5p在其中显著富集，初步证据提示其可通过抑制MAP4K3磷酸化转录因子EB(TFEB)调控自噬流活化。据此，本项目拟利用免疫共沉淀、RNA干扰和激光共聚焦等方法深入探讨MSC外泌体Let-7b-5p通过MAP4K3/TFEB信号通路介导自噬流活化进而抑制ACLF过程中肝细胞焦亡的病理生理学意义与分子机制，以期为优化MSC的治疗方案提供新策略。

Our previous studies have shown that mesenchymal stem cells (MSC) can improve the outcome of acute-on-chronic liver failure (ACLF), however the mechanism is still unknown. In our pre-experimental results indicate that MSC and its exosomes components could inhibit ACLF mice hepatocyte pyroptosis, induce autophagic flux activation, and significantly up-regulate hepatocyte pyroptosis after blocking autophagic flux activation. After inhibiting the secretion of MSC exosomes, the activation of hepatocyte autophagy flux was obviously blocked, and cell pyroptosis increased. Further analysis of microRNA sequencing showed that Let-7b-5p was enriched in MSC exosomes and mediated autophagic flux activation by inhibiting MAP4K3 phosphorylated transcription factor EB(TFEB). Thus this project would use co-immunoprecipitation, RNA interference and laser confocal methods to explore the mechanism of MSC inhibits hepatocytes pyroptosis through activating autophagic flux, which was supposed to via its exosomes derived Let-7b-5p and regulate the MAP4K3/TFEB signaling pathway, in order to provide a new strategy for the optimization of MSC treatment for ACLF.