TIMP-1/miR145/CCL2轴通过肝星状细胞分泌的外泌小体调控肝脏巨噬细胞趋化的作用机制研究

The activation of hepatic stellate cells (HSCs) and the recruitment of hepatic macrophages to the injured liver are the central events in the progression of liver fibrosis. The tissue inhibitor of metalloproteinase-1 (TIMP-1) secreted by HSCs plays crucial role in fibrogenesis. Our previous studies found that the antifibrotic effects of a recombinant adeno-associated virus carrying small interfering RNA targeting TIMP-1 in rat liver fibrosis were associated with decreased recruitement of hepatic macrophages and less expression of C-C motif ligand 2 (CCL2) in the liver. However, the mechanisms remain unclear. Exosomes, small vesicles containing miroRNAs (miRNAs) and other nucleic acids in the form of steady state, are important in intercellular communication. Our preliminary data has also shown that the changes of miRNA in exosomes isolated from cell cultrue medium were consistent with that in the cultured cells, indicating that the information carried by exosomes reveals the cell conditions. Based on our preliminary data that miRNA145 maybe correlated with the transcription of CCL2, in the present study we will further determine the regulatory mechanism of TIMP-1 on CCL2 transcription through miRNA145. Furthermore, we will elucidate the intercellular communication by exosomes containing miRNA145 between HSCs, as well as HSCs and hepatic macrophages and expression of CCL2 in target cells. Lastly, we will investigate whether the infusion of exosomes containing miRNA145 will alter CCL2 expression in serum and liver of receptor mice and have antifibrotic effectes in carbon tetrachloride induced liver fibrosis mice model. This study may help clarify the regulating mechanism of TIMP-1 on CCL2 transcription and feasibility of exosomes as vector in antifibrotic strategy.

肝星状细胞活化和肝脏巨噬细胞的募集是肝纤维化过程中重要环节，星状细胞分泌的TIMP-1有促肝纤维化作用。我们前期研究发现：动物体内干预TIMP-1具有的抗肝纤维化作用，与巨噬细胞向肝脏的募集减少以及CCL2分泌降低相关，但作用机制尚不明确。同时发现细胞分泌的外泌小体可以稳定传递miRNA信息，反映细胞功能状态。本课题拟首先以前期发现的与CCL2表达相关的miRNA145为切入点，明确TIMP-1通过miRNA145调控CCL2蛋白表达的分子机制。其次观察携带miRNA145的外泌小体在星状细胞之间以及星状细胞和巨噬细胞之间的胞间传递及对CCL2蛋白表达的影响。最后利用小鼠四氯化碳肝纤维化模型，探讨TIMP-1下调后的星状细胞通过外泌小体传递抑制巨噬细胞趋化信息进而发挥抗肝纤维化作用的可能性。本研究将明确TIMP-1调控CCL2的具体分子机制并为外泌小体为载体的抗肝纤维化治疗提供实验室依据。

我们的前期研究发现，靶向大鼠基质金属蛋白酶抑制剂（TIMP）-1基因的小干扰RNA的重组腺相关病毒（rAAV/siRNA-TIMP-1），通过调控基质金属蛋白酶活性和活化肝星状细胞（HSC）数量，发挥有效的抗肝纤维化作用。同时，我们也观察到通过rAAV/siRNA-TIMP-1的干预，肝脏巨噬细胞的数量大大减少。因而本课题进行了下调TIMP-1可以抑制巨噬细胞募集的机制探讨。我们首先以siRNA-TIMP-1预防性治疗大鼠和小鼠CCl4肝纤维化，观察到siRNA-TIMP-1不仅可以减轻肝纤维程度，同时有效降低了肝组织中单核细胞趋化蛋白（MCP）-1及其上游转录因子Fli-1的表达。随后，在对大鼠肝星状细胞系HSC-T6和小鼠原代肝星状细胞进行siRNA-TIMP-1干扰后，发现不仅两种细胞中TIMP-1表达下将，同时伴有MCP-1表达降低及对巨噬细胞的募集作用减弱。siRNA-Fli-1转染不仅降低HSCs中Fli-1的表达，其下游分子MCP-1表达相应降低；HSCs中转染Fli-1过表达质粒后，MCP-1表达升高。转染miRNA145-mimics后，HSCs中Fli-1和MCP-1表达降低；转染miRNA145-inhibitor后，HSCs中Fli-1和MCP-1表达升高。通过荧光素酶报告基因检测，证实miRNA145对Fli-1的3’UTR存在至少两个结合位点，可以调控Fli-1表达。由此，我们证实了TIMP-1可以通过miRNA145调控转录因子Fli-1的表达，进而影响MCP-1表达而发挥对巨噬细胞的募集作用。.随后，我们进一步探究外泌体是否作为细胞间重要传递介质在肝纤维化中发挥作用。我们首先证实了HSC来源的外泌体可以被HSC和巨噬细胞有效捕获；给予外泌体抑制剂GW4869处理后，即使通过siRNA-TIMP-1有效抑制了HSC中MCP-1的表达，但其对巨噬细胞的迁移抑制作用减轻。随后，我们用正常小鼠血清中提取的外泌体输入CCl4和TAA诱导的肝纤维化小鼠体内，发现正常小鼠血清来源的外泌体可以有效减轻CCl4和TAA诱导的肝纤维化程度，且血清来源的外泌体主要靶向肝细胞和HSC。最后，为提高载体的丰度，我们以生物膜载体代替外泌体，观察其在小鼠体内的靶向性。与脂质体相比，生物膜载体可以有效靶向肝脏HSC,为今后以HSC为治疗靶点的药物运输提供了有力支持。