基于Wnt/β-catenin的具有青光眼视神经保护作用中药高通量筛选体系的建立及验证

The existing methods for the treatment of glaucomatous optic nerve are not ideal, and the emergence of innovative drugs is urgently needed. High throughput screening system has gradually become the main method of drug screening. The key to the establishment of the screening system is the drug target. The key signal pathway of retinal ganglion cell apoptosis in glaucoma is Wnt/ beta -catenin, the key drug target is Pax6 (molecular target) and RGCs (cell target). From this point of view, genetic engineering and transgenic technology will be used to establish a high throughput screening system for the protective effect of glaucomatous optic nerve. The establishment of the system will be provided as key technologies for high-throughput screening of traditional Chinese medicine components of the optic nerve protective effect for glaucoma.High throughput screening of traditional Chinese medicine components is beneficial to the research and development of new drugs..Previous studies showed that QingGuangAn II has a good protective effect on the optic nerve in glaucoma state, could reduce the apoptosis of glaucoma RGCs. In order to verify the established screening system, QingGuangAn II Chinese medicine library will be prepared in this study.The effective components will be screened by the system, and the screening results will be verified for animal models. If the verification is successful, we can not only establish practical screening system , but also find effective components of QingGuangAn II for optic nerve protective effect on glaucoma, which is helpful for the innovation of drug discovery.

现有对青光眼视神经保护的治疗方法并不理想，亟需创新药物出现。高通量筛选体系已逐渐成为药物筛选的主要方法。筛选体系建立的要点在于药靶。青光眼视网膜神经节细胞凋亡的关键信号通路是Wnt/β-catenin，关键药靶是Pax6（分子靶标）和RGCs（细胞靶标）。本研究从此入手，采用基因工程技术和转基因技术，建立青光眼视神经保护作用中药的高通量筛选体系。体系的建立，将为青光眼视神经保护作用的药物筛选提供关键共性技术，利用该体系对中药组分库进行高通量筛选，有利于新药研发。.前期研究表明青光安Ⅱ号方对青光眼状态下视神经有良好的保护作用，可以减少青光眼RGCs的凋亡。为了验证所建立的筛选体系，本研究将制备青光安Ⅱ号中药组分库，利用该体系进行有效组分筛选，并将筛选结果进行动物模型验证。若验证成功，不仅能证明所建立的筛选体系的实用性，也能找出青光安Ⅱ号对青光眼视神经保护作用的有效组分，有助于创新药物的发现。

迄今为止，人们也还尚未找到一种十分有效的青光眼视神经保护中药，其中一个重要原因就是缺乏青光眼视神经保护中药高效筛选体系。.青光眼视神经病变的病理基础在于视网膜神经节细胞（RGCs）的渐进性凋亡和轴突的丢失。Pax6基因在青光眼视神经病变中起重要作用，Pax6在RGCs及视网膜组织上的适度表达，有利于抑制细胞凋亡，保护视神经，同时Pax6基因又是Wnt /β-Catenin信号通路的一个靶位。利用Pax6基因转录相当于Wnt/β-Catenin信号通路激活的这一特点，建立良好的Pax6活性监测系统以反映外源性物质对Wnt/β-Catenin信号通路的促进效果，可以用来进行青光眼视神经保护药物筛选。.本研究从此入手，采用基因工程技术和转基因技术，建立了基于Wnt/β-Catenin/Pax6的具有青光眼视神经保护作用中药高通量筛选体系。体系的成功建立，为青光眼视神经保护药物筛选提供了关键共性技术。 .制备溶剂分离联合大孔树脂分离的方法建立了青光安II号方中药组分库，利用该体系对中药组分库进行高通量筛选。筛选出有效组份1-4、2、2-1及2-4，并将筛选组分2进行动物模型验证。 .动物模型验证结果显示，青光安II号方中药组分2高浓度组能明显改善青光眼视网膜结构，抑制RGCs凋亡，抑制GSK-3β蛋白和促进β-Catenin、Pax6 蛋白在视网膜中的表达。