miR-188-5p调控骨髓基质干细胞参与脉络膜新生血管发生发展的作用及机制研究

Choroidal neovascularization (CNV) is a common cause of severe and irreversible visual loss. We firstly reported that bone marrow-derived mesenchymal stem cells (MSCs) could be specifically recruited into CNV lesions, thus, MSCs could serve as delivery vehicles of therapeutic agents for targeted treatment of CNV. However, MSCs also contribute to the development of CNV, which hinder the clinical use of MSCs for CNV treatment. To accomplish this, it is required to expand our understanding of the mechanism of MSCs contribution to CNV.We found previously that hypoxia significately down-regulated the expression of miR-188-5p in MSCs, and the target genes of miR-188-5p might be matrix metalloproteinases (MMPs)-2 and MMP-13. MMPs is necessary for CNV formation. It is reported that MMP-13 promotes CNV development via MSCs. Moreover,the most powerful endogenous inhibitor of CNV,pigment epithelium derived factor (PEDF), is the substrate of MMP-2. Therefore, we propose that miR-188-5p is the critical regulator of MSCs contribution to CNV development, hereby, miR-188-5p regulates degradation of extracellular matrix and PEDF in CNV, and in turn affects the prolification, migration and apoptosis of vacular cells and fibrosis of CNV via redulating of MMP-2/ -13 expression. This project is going to testify this hypothesis through luciferase report system, lentivirus transduction, fluorescent image in vivo and so forth, aim to promote the application of MSCs for CNV treatment.

脉络膜新生血管(CNV)常造成严重视力损害。我们首先报道骨髓基质干细胞(MSCs)向CNV特异性趋化，可作为载体靶向释放药物。但同时MSCs也参与CNV生成，是一把"双刃剑"。需阐明其参与机制方能"趋利避害"。我们前期研究发现缺氧显著下调MSCs中miR-188-5p表达，其靶基因可能是基质金属蛋白酶(MMPs)-2和-13。MMPs是CNV生成必需因子。据报道MMP-13促CNV发展作用主要通过MSCs介导；MMP-2降解CNV最强内源抑制剂－色素上皮衍生因子(PEDF)，强烈提示miR-188-5p是MSCs参与CNV机制中的关键节点分子：miR-188-5p调控MMP-2/-13表达，影响CNV细胞外基质和PEDF降解，进而影响血管细胞出芽、移行、凋亡及CNV纤维化。本项目拟通过荧光素酶报告系统、慢病毒过表达/干涉技术、活体荧光成像等方法论证该假说，为CNV治疗提供新思路和靶点。

我们前期证实骨髓基质干细胞（MSCs）向CNV特异性趋化，可作为载体靶向释放药物。但同时MSCs也参与CNV生成，是一把“双刃剑”。阐明其参与机制方能“趋利避害”。我们通过基因芯片筛选发现缺氧显著下调MSCs中miR-188-5p表达，其靶基因可能是MMPs-2和-13（MMP-2/MMP-13）。而MSCs是BMCs的重要组成部分，其分泌的MMPs是CNV生成时BMCs侵入和原位细胞移行所必需的因子。因此，我们欲探讨miR-188-5p对BMCs/MSCs中MMP-2/MMP-13表达和CNV生成的影响。我们检测了缺氧MSCs及活体CNV内及CNV内BMCs的miR-188-5p和MMP-2/MMP-13的表达，证实miR-188-5p和MMP-2/MMP-13表达规律，发现MMP-2/MMP-13的表达随时间动态变化，BMCs对其表达的迅速上调起主要作用。CNV中BMCs内miR188-5p与MMP-2/MMP-13表达变化呈负相关，提示miR-188-5p对MMP-2/MMP-13表达存在调控作用。双荧光素报告研究证明了miR188-5p对MMP-2/MMP-13表达的直接调控。我们制备了mimics和inhibitors作为体外调节miRNA水平的工具，证实MMP-2/MMP-13是miR-188-5p的靶基因以及miR-188-5p对其调控作用。在体内，我们采用高效miRNA模拟物agomir在眼局部过表达miR-188-5p，减少BMCs分泌MMP-2/MMP-13，抑制BMCs侵入CNV和分泌促血管生成因子，减少PEDF的降解，成功抑制CNV的发生发展。最终证实了通过miRNA对CNV进行高效靶向治疗的可行性。综上所述，本项目证实了miR-188-5p调控BMCs中MMP-2/-13表达，影响CNV中细胞外基质和PEDF降解，进而影响BMCs参与CNV和CNV内的促血管生成因子水平，利用这一机制，实现单一靶点干预即可达到瀑布式效应、高效治疗CNV的目的。本项目从全新角度探索了BMCs参与CNV发生的机制及miRNA结合BMCs用于CNV靶向治疗的潜能，为CNV相关眼病的治疗提供了新思路和靶点。