人脐带间充质干细胞外泌体通过miR-326抑制Smo受体活性逆转大鼠肝纤维化的研究

Currently, it is thought that MSCs perform their functions primarily through their paracrine role rather than cell replacement. And in Paracrine system, miRNAs of exosomes (Exo) play a crucial role in liver injury. In our previous study, we found that the human umbilical cord MSCs exosomes (huc-MSCs-Exo) could significantly reduce the degree of liver fibrosis in rats. However，compared with other types of MSCs, higher level of miR-326 was detected in huc-MSCs-Exo. Further experiments showed that miR-326 level was significantly increased and the target gene α-SMA was decreased in hepatic stellate cell line LX2 co-culturing with huc-MSCs-Exo in vitro. It suggests that miR-326 plays a key role in reversing hepatic fibrosis. Therefore, through injecting nano-iron labeled huc-MSCs-Exo into rat livers with hepatic fibrosis in situ, we analyzed the target cells of exosomes in injury liver by magnetic resonance imaging and investigated the effect of huc-MSCs-Exo on Hedgehog signaling pathway in rat fibrosis liver. And we established a co-culture model in vitro to clarify the key targets and mechanism of miR-326 in regulating hepatic stellate cells. These results can provide theoretical basis and drug targets for clinical transformation of exosomes in treatment of liver diseases.

目前认为间充质干细胞（MSCs）主要是通过旁分泌作用而不是细胞替代发挥作用。而旁分泌系统中，外泌体（Exo）中的微小RNA（miR）在肝损伤中发挥重要作用。课题前期研究发现人脐带（hUC）MSCs-Exo显著减轻大鼠肝纤维化。进一步研究发现，相比其他MSCs-Exo，huc-MSCs-Exo中存在高水平的miR-326。而huc-MSCs-Exo与肝星状细胞系LX2共培养后，LX2中miR-326水平显著升高，并且纤维化标志蛋白α-SMA表达降低，提示miR-326可能是逆转肝纤维化的关键分子。因此本研究拟通过纳米铁剂标记huc-MSCs-Exo，利用磁共振成像分析外泌体在纤维化肝脏的主要靶细胞，明确huc-MSCs-Exo对大鼠肝纤维化Hedgehog信号通路的影响；并在体外建立共培养模型，探讨miR-326调控肝星状细胞的关键靶点和机制。为外泌体治疗肝损伤疾病提供理论依据和药物靶标。

本项目的研究背景是人脐带间充质干细胞外泌体在CCl4损伤的小鼠肝纤维化模型中，通过 miR-326失活肝星状细胞的Hh信号通路中的Smo受体，减少Hh反应性细胞，抑制肝纤维化的发生发展。项目通过研究huc-MSCs-Exo对小鼠肝纤维化的修复作用和对Hh信号通路的影响，研究huc-MSCs-Exo在肝纤维化的小鼠肝脏中主要的靶细胞以及外泌体中miR-326 调控HSCs的Hh信号通路的靶点和机制研究。结果表明huc-MSCs-Exo在小鼠肝纤维化的肝脏中主要的靶细胞是肝星状细胞，项目采用的纳米铁示踪剂标记的huc-MSCs不仅在小鼠体内验证了它的安全性和可示踪性，并且在大动物比格犬的脊髓损伤模型中进行了验证。外泌体高表达的miR326作用在肝星状细胞的Hh通路的Smoothened蛋白，抑制Gli家族转录因子的活化和核定位，Hh信号通路被抑制，抑制静止的肝星状向活化的HSCs转变，减轻肝纤维化程度。项目组同时发展了一种微创开流微灌注(OFM)技术，将huc-MSCs原位移植到肝脏，并对CCl4诱导的肝纤维化小鼠的肝脏微环境进行活体监测，与静脉注射相比，OFM途径可增加huc-MSCs在肝脏中的积聚，并能更有效地改善肝组织结构重塑，减少纤维化肝脏胶原沉积。