脂肪组织外泌体调控Hippo-YAP通路促进皮肤创面缺损修复机制研究

The cutaneous wound healing caused by burns, diabetic foot and other diseases is a clinically urgent problem to be solved. Exosomes can promote tissue regeneration through the involvement of substances such as miRNAs and proteins in the transduction and exchange of signals between cells. However, it is still unclear how to repair skin defects and their regulatory mechanisms by using exosomes of the adipose tissue (E-AT). Studies have shown that Hippo-YAP pathway plays an important role in cell proliferation, apoptosis and tissue regeneration. Earlier studies found that E-AT can regulate a variety of signaling pathways to promote vascular and soft tissue regeneration, and some high expression of miRNA can down regulation YAP, LATS2 and other core components on the Hippo-YAP pathway. Therefore, this project intends to study the effects of E-AT on the key steps of revascularization, collagen synthesis and re-epithelization in the skin defects healing in rats. At the same time, it is revealed that E-AT can promote the proliferation, migration and function of the relational cells during wound healing. Finally, it will be the first time to further investigate whether LATS2 targets miR-92a-3p and to verify its ability to regulate this pathway. This study laid the theoretical and experimental foundation for the clinical application of E-AT to repair skin and soft tissue defects and enrich the mechanism of tissue regeneration by miRNA.

因烧伤、糖尿病足等造成的皮肤软组织缺损修复是临床上急需解决的问题。外泌体能够通过miRNA及蛋白质等物质参与细胞之间的物质转导和信号交流促进组织再生，但是使用脂肪组织外泌体（E-AT）来修复皮肤缺损及其调控机制尚不清楚。研究证明Hippo-YAP通路在细胞增殖凋亡及组织再生上发挥重要作用。本项目前期发现E-AT能够调节多种信号通路促进血管及软组织再生，且部分高表达miRNA能够下调YAP、LATS2等核心组件作用于Hippo-YAP通路。因此，本项目拟研究E-AT对大鼠皮肤缺损愈合中血管再生、胶原合成及再上皮化等关键步骤的影响。同时，揭示E-AT促进创面愈合过程中相关细胞的增殖、迁移及功能的调控机制。最后，将首次深入研究LATS2是否为miR-92a-3p的靶标，并验证其调控该通路的能力。该研究为临床应用E-AT修复皮肤软组织缺损及丰富由miRNA参与的组织再生机制奠定理论及实验基础。

因烧伤、糖尿病足等造成的皮肤软组织缺损修复是临床上急需解决的问题。外泌体能够通过miRNA及蛋白质等物质参与细胞之间的物质转导和信号交流促进组织再生，但是使用脂肪组织外泌体来修复皮肤缺损及其调控机制尚不清楚。本项目发现脂肪外泌体能够促进角质形成细胞增殖与迁移能力，同时也促进真皮成纤维细胞增殖及胶原沉积。这两种细胞的活化与增殖与创面愈合密切相关。随着多年来的研究发现，miRNA 对靶基因的基本调控机制已被研究得较为透彻，其调控创面缺损修复⽅面的作用也有相当的⽂献支持，特别是对成纤维细胞及角质形成细胞功能⽅面的研究。而外泌体主要是通过其囊内携带的miRNA 进⾏细胞间物质转导及信息交流。鉴于前期表达芯片结果显示脂肪来源外泌体中多种mirna高表达，因此本研究重点关注脂肪外泌体中高表达的miR-92a在角质细胞及成纤维细胞的细胞功能的影响。结果发现，miR-92a过表达促进了细胞增殖，迁移和胶原沉积。而miR-92a inhibitor 能够逆转外泌体对上述两种细胞的作用。我们进一步通过在线预测工具发现miR-92a与LATS2的3’UTR区存在结合位点。作为Hippo-YAP通路的核心组件，活化的Lats1/2 主要的靶蛋白为转录因⼦TAZ/YAP，磷酸化的TAZ/YAP 与14-3-3 蛋白结合后滞留于胞浆中⽽⽆法进⼊细胞核。当磷酸化通路被抑制后，未磷酸化的TAZ/YAP 进⼊细胞核与TEAD 等转录因⼦结合调控细胞增殖，分化及迁移。通过荧光素酶报告基因实验，验证了LATS2为miR- 92a-3p的靶标，并通过过表达及敲低转染技术，证实了miR-92a/LATS2 轴在调控角质形成细胞及真皮成纤维细胞增殖及活化中的作用。最后，通过构建大鼠皮肤创面模型，在体研究了脂肪来源外泌体对伤口愈合的作用，并且免疫组化染色明确了LATS2/YAP/ZAP通路的活化在伤口愈合中存在动态变化。该研究为临床应用脂肪来源外泌体修复皮肤软组织缺损及丰富由miRNA参与的组织再生机制奠定理论及实验基础。并且提示靶向Hippo-YAP通路的治疗策略具有应用潜力。