脂肪干细胞外泌体源的miRNA-19通过“P47phox-ROS”通路调节肝衰竭大鼠肝组织炎症的机制研究

我们前期发现干细胞来源的外泌体可有效抑制肝衰竭时肝脏的炎性损伤，二代RNA测序结果提示hASCs外泌体治疗组大鼠肝组织中与炎症、趋化信号相关的通路基因显著下调，如：趋化因子-P47phox-ROS。我们对外泌体内microRNA进行高通量测序，发现其内多种具有抑炎作用的miRNAs与此通路变化显著相关。体外实验发现与其他miRNAs相比，miR-19b-3p可显著抑制巨噬细胞活化。通过targetscan生物信息预测，我们发现miR-19恰好可以与NADPH氧化酶的亚基p47phox结合。综上所述，我们提出以下科学假说：人hASCs外泌体到达大鼠肝组织后释放出miRNA-19，抑制P47phox-ROS通路和活化巨噬细胞介导的炎症性损伤，提高其生存率。我们拟在本课题中通过构建沉默或过表达miRNA-19的慢病毒，并运用实时定量PCR、荧光素酶报告基因和RIP等技术，阐明这一机制。

We found that the exosomes derived from stem cells can effectively inhibit the inflammatory damage of liver during liver failure. The results of RNA sequencing suggested that the pathway genes related to inflammation and chemotaxis in the liver tissue of rats in the hASCs exosomes treatment group are significantly down regulated, such as chemokine-p47phox-ros. We sequenced microRNAs in vitro, and found that a variety of miRNAs with anti-inflammatory effect were significantly related to the change of this pathway. Compared with other miRNAs, mir-19b-3p significantly inhibited macrophage activation in vitro. Through the biological prediction of targetscan software, we found that Mir-19 could combine with P47phox, a subunit of NADPH oxidase. To sum up, we propose the following scientific hypothesis: the human hASCs exosomes release miRNA-19 after reaching rat liver tissue, inhibit p47phox-ROS pathway and activate macrophage mediated inflammatory damage, and improve its survival rate. In this project, we intend to construct a silent or over expressed miRNA-19 lentivirus, and use real-time quantitative PCR, luciferase reporter gene and rip technology to clarify its specific mechanism.