靶向调控miR-146a表达重建巨噬细胞M1/M2极化平衡对脉络膜新生血管的阻遏效应及机制研究

Choroidal neovascularization (CNV) is the key pathogenesis of irreversible blindness in the elderly people. Recently, increasing evidence supports that different sub-types of macrophages have different effects on the CNV formation: M1 polarized macrophages inhibit the formation of CNV while M2 polarized macrophages promote CNV formation; In additon, the pathologic balance of M1Low/M2High is an important trigger for CNV formation at the early stage. Our previous study showed that the endogenous expression of microRNA-146a (miR-146a) was a key regulator for the polarization of macrophages and targeted regulation of the miR-146a expression in the macrophages could precisely controlled the M1 or M2 polarization of macrophages. Based on the above facts, we suppose that targeted regulation of the miR-146a expression in the macrophages could transform the balance of polarization from M1Low/M2High to M1High/M2Low, which should have an inhibitive effect on the development of CNV. In this research, we are going to reconstruct the balance of M1/M2 polarized macrophages in the focal lesion through regulating the endogenous expression of miR-146a in the macrophages using the CRISPR interference (CRISPRi) and CRISPR activation (CRISPR a) technology and demonstrate whether it inhibit the CNV formation through regulating the inflammatory micro-environment and the expression of vascular endothelial growth factor (VEGF), which would provide a new therapeutic strategy for the prevention and treatment of CNV related diseases.

脉络膜新生血管（CNV）是造成老年人不可逆性盲的重要病因。不同亚型的巨噬细胞对CNV的作用不同：M1巨噬细胞抑制CNV而M2巨噬细胞则促进CNV的形成，且早期病灶中M1Low/M2High极化平衡的建立被证实是诱发CNV形成的早期重要生物学事件。我们的前期研究发现miR-146a是巨噬细胞极化方向的重要调控开关，靶向调控miR-146a表达可对巨噬细胞M1或M2极化方向进行精确调控。因此提出科学假说：通过靶向调控巨噬细胞内源性miR-146a表达可将病理微环境中促进CNV形成的M1Low/M2High极化平衡逆转为抑制CNV形成的M1High/M2Low极化平衡，有望抑制CNV形成。本研究拟采用CRISPRi/a技术靶向调控miR-146a表达来重建病灶中巨噬细胞M1/M2极化平衡，通过改变病灶炎症微环境和VEGF表达而发挥对CNV的阻遏效应并阐明其机制，为防治CNV提供一条崭新思路。

本项目通过QPCR、WB、ELISA和双荧光素酶报告基因等实验发现，在细胞水平，靶向下调巨噬细胞内miR-146a水平可抑制巨噬细胞向M2型极化，上调miR-146a可促进巨噬细胞向M2型极化，并且下调miR-146a可抑制巨噬细胞分泌VEGF，从而抑制内皮细胞的增殖、迁移和管腔形成。另外下调miR-146a可抑制NF-κB通路的活性，通过TargetScan数据库预测筛选和双荧光素酶报告基因实验验证发现，miR-146a与OTUD7B的3'UTR区域存在结合位点，miR-146a可能通过miR-146a-OTUD7B-NF-κB调控轴发挥作用。在动物水平发现，下调miR-146a可以有效抑制小鼠CNV病灶的形成和渗漏，并且下调miR-146a同样可以降低RPE/脉络膜组织的VEGF和ICAM1表达量，同时抑制NF-κB通路的活性。HE切片和ERG实验发现玻璃体腔注射miR-146a抑制剂不会对小鼠视网膜的结构和功能产生毒性。所以，下调miR-146a有望成为治疗CNV的新方向，值得深入探究。项目资助发表SCI论文一篇，中文核心一篇，待发表SCI论文两篇，培养博士研究生1名，硕士研究生3名。