间质干细胞来源exosome抗肝纤维化作用机制研究

There are still no effective antifibrotics available and mesenchymal stem cells (MSCs) transplantation has recently gained increasing attention in liver fibrosis. Our preliminary study suggested that MSC derived exosome(MSC-exosome), an active component secreted by MSCs, could ameliorate carbon tetrachloride induced liver fibrosis in mice. However, the mechanisms for this inhibition remain unknown. Activation of hepatic stellate cells (HSCs) is the key event in liver fibrosis, whereas previous studies have demonstrated an important role of microRNAs during the activation of HSCs. We therefore hypothesize that exosome-mediated transfer of microRNAs play a critical role in MSC-exosome's inhibition of HSCs activation in liver fibrosis. To test the hypothesis, my research will focus on the following three specific aims: Aim 1) Determine the role of MSC-exosome in HSCs activation through in vivo and in vitro experiments.Aim 2) Identify the key microRNAs roled in HSCs activation by using microRNA microarray for microRNAs screening and microRNA modified MSC-exosome for microRNA activity assay.Aim 3) Determine the target genes and pathways of the key microRNAs involved in HSCs activation,which will be predicted through microRNA target gene databases? and verified by using various molecular biological approaches including Luciferase Reporter Gene Assay, Western blot, quantitative real-time PCR. We expect that the results obtained from proposed studies will help us to understand how MSC-exosome works in liver fibrosis, which could lead to improved therapy design and provide the molecular basis for the development of new therapeutic methods for liver fibrosis. Thus, it is of great value and clinical application prospects.

肝纤维化治疗目前尚缺乏很好的方法，间质干细胞（MSCs）移植在肝纤维化治疗中的作用越来越引起人们的重视。我们初步研究发现脐带MSC来源的囊泡（MSC-exosome）作为脐带MSCs旁分泌的活性成分能够抑制四氯化碳诱导的小鼠肝纤维化，然而其抑制作用机制并不清楚。为此，本课题以MSC-exosome在肝纤维化中向肝星状细胞转移的microRNAs为切入点，体内外观察MSC-exosome对肝星状细胞活化的抑制作用；采用microRNA芯片筛选MSC-exosome向肝星状细胞转移的microRNAs；明确MSC-exosome抑制肝星状细胞活化的关键miRNAs；寻找关键miRNAs调控的肝星状细胞活化相关靶基因及信号传导通路，阐明MSC-exosome修复肝纤维化的作用机制。本研究将为MSC-exosome治疗肝纤维化提供新的思路和实验依据，具有十分重要的价值和临床应用前景。

间质干细胞（MSC）来源的囊泡 (MSC-exosome)是MSC旁分泌的有效活性成分。MSC在肝损伤修复中应用已有相关报道，但有关MSC-exosome修复肝纤维化及其机制的研究尚未见报道。肝星状细胞（HSC）活化分泌大量胶原是肝纤维化发生的重要机制。因此，本研究在成功分离脐带MSC-exosome并初步证实其抗纤维化作用的基础上，进一步观察了肝纤维化修复过程中脐带MSC-exosome对HSC活化的抑制作用；探讨脐带MSC-exosome修复肝纤维化的作用及机制。根据计划书研究内容，课题组培养脐带MSC，分离纯化获得了脐带MSC-exosome，并进行了鉴定。在此基础上完成了以下内容：1）脐带MSC-exosome在肝细胞内定位及对HSC活化的抑制作用。 2）体内移植MSC-exosome到纤维化小鼠肝组织，明确了MSC- exosome在肝组织内定位，检测MSC- exosome对肝纤维化和肝组织胶原的抑制作用。3) 采用生物信息学方法预测，并经定量PCR检测获得相关miRNAs（Let7b，Let7c），确定MSC-exosome转移的miRNAs中Let7b是具有抑制HSC活化能力的关键分子之一。同时，我们根据计划书对部分内容作了适当的调整，采用液相蛋白芯片对脐带MSC-ex中活性蛋白进行了检测，并发现肝纤维化抑制相关蛋白14-3-3ζ、谷胱苷肽过氧化物酶-1（Gpx1）。明确MSC-exosome转移的14-3-3ζ、Gpx1是MSC-exosome中具有抑制HSC活化能力的关键分子。本研究成功观察了脐带MSC-exosome对HSC活化的抑制作用，证实了Let7b、Gpx1、14-3-3ζ是调控HSC活化和胶原分泌的关键。本研究成功观察了脐带MSC-exosome对HSC活化的抑制作用，证实了Let7b、Gpx1、14-3-3ζ是调控HSC活化和胶原分泌的关键分子,初步阐明了MSC-exosome修复肝纤维化的机制。本研究将为MSC-exosome治疗肝纤维化提供新的思路和实验依据，具有十分重要的价值和临床应用前景。