AAV2-miRNA-21靶向增强小梁网房水流出的作用及其机制研究

Glaucoma has become first irreversible blinding disease in the world. The elevation of intraocular pressure (IOP) is the most important risk factor. And the main pathogenesis is the increased outflow resistance. IOP reduction is the only proven effective treatment at present. However, these drugs all function by single pathway and rarely directly target Schlemm's canal. Therefore, it is often necessary to combine and repeatedly use drugs to reduce IOP effectively. It's urgent to find new drugs. In our previous work, the expression of miRNA-21 in porcine senescent Schlemm canal(SC) endothelial cells decreased significantly. Overexpression of miR-21 can increase the permeability of porcine SC endothelial cells. By injecting it into the anterior chamber of the mouse, the IOP was significantly reduced (more than 20%), and the duration was over 48h. It is suggested that mir-21 could be an effective molecular for lowering IOP. This study intend to further investigate the mechanisms of miR-21 multiple targeting function in regulation of IOP by porcine Schlemm's canal endothelial cell and wild type/eNOS knockout mouse. And then study the long-term effectiveness of miR-21 in sGCα1 defect mice model (POAG model), by targeted transfection to trabecular meshwork endothelial cells and SC endothelial cells carried by Anc80L65 AAV2.

青光眼是全球首位不可逆致盲眼病，眼压升高是最重要危险因素，主要病理机制为小梁网流出受阻。降眼压治疗是目前唯一有效的治疗方法，其中以药物降压最为安全。然而，目前临床降眼压药物均为单一靶点，并且鲜有以调控小梁网流出为机理的药物，因此往往需联合、反复用药才可有效降压。迫切需要探索针对发病机制的新药物。我们前期工作中发现衰老Schlemm管（SC）内皮细胞miRNA-21表达显著下降。过表达miR-21可以增加SC内皮细胞通透性。将其注入小鼠前房能够显著降低眼压（大于20%），持续时间超过48h。提示miR-21可望成为有效的降压靶点。为此，本研究拟以猪房水静脉丛细胞、eNOS敲除小鼠为研究对象，深入研究miR-21降低眼压的多靶点分子机制，进而以sGCα1缺陷小鼠（POAG模型）为对象，以靶向性转染小梁网及SC内皮细胞的Anc80L65 AAV2携带miR-21，研究miR-21的长效降压作用。

目前对于原发性开角型青光眼发病机制的探索，仍在不断进行中。小梁网途径是调节眼内压的重要调节区域，针对于小梁网途径的房水流出调节机制与药物仍在不断开发，是青光眼降眼压治疗中的重要研究领域。.Schlemm（SC）管内皮细胞是小梁网流出途径阻力最大的位置，课题组发现了小分子核酸miR-21-5p促进房水静脉细胞通透性增加，显著提高小鼠眼球内小梁网房水流出率、降低眼内压，并且其调节作用与多项调节细胞通透性、细胞外基质重塑和细胞骨架相关的信号通路相关，并且在原发性开角型青光眼患者房水中，miR-21-5p有一定趋势的降低，提示miR-21-5p可能是调控眼内压的靶点。.基于项目前期工作经验，课题组探索了作用于小梁网途径的核酸载体：AAV-DJ（腺相关病毒DJ型）与聚多巴胺-聚乙烯亚胺纳米颗粒（PDA/PEI NPs）在体转染的安全性可行性，并评估了用于载入目的核酸降低眼压的有效性，为针对小梁网途径的在体核酸转染提供了参考方案。.除上述探索外，项目组进一步通过对青光眼细胞模型的全转录组学联合分析，进一步寻找与小梁网途径流出通道阻力相关的重要分子与机制，聚焦调节SC 生物力学的靶点。此外，对于影响小梁网途径发育于维持的重要信号通路也进行了相关探索，并在国人的临床样本中多角度验证了该通路基因突变的致病性。.综上，本项目探索了在小梁网房水流出途径中的调节功能与机制的重要分子，以及作用与小梁网房水流出通路的核酸载体，对于针对小梁网途径的机制研究和治疗策略提供更丰富的信息和参考。本项目已发表并标注本课题的SCI论文共11篇，国内核心期刊6篇，获得国家发明专利授权2项。