肝郁证慢性高眼压大鼠视网膜神经节细胞miRNAs表达特征及疏肝通窍法对其表达的影响

Glaucoma is high blinding eye disease, has the obvious physical characteristics， according to syndrome differentiation of traditional Chinese medicine, liver depression syndrome of glaucoma is the most common type. Existed research shows that about 30% of protein-coding genes are regulated by miRNAs, suggest physical constitution is determined by genes. Studies have found that in the retina and other ocular tissues, the expression of miRNAs have tissue specificity and developmental stage specificity, it suggests that miRNAs have potentially functional specificity in the retina and other ocular tissues and cells. This research is based on TCM viscera-state theory, use TQMM NO.4 decoction as intervention, take optic ganglion cells (RGCs) in rats of miRNAs as the research target, adopts the miRNA chip hybridization, RT-PCR detection, P-VEP and cell ultrastructure as detection methods, respectively from two directions study the pathogenesis of physical constitution of liver depression syndrome glaucoma, curative effect and correlation of free liver Qi and open orifice method regulation genes transcription in liver depression syndrome glaucoma; through screening differentially expressed of miRNAs in RGCs after Chinese medicine intervention, predict and verify its' target genes. From Holism, gene regulation and cell layers, study the possible mechanism of liver depression syndrome glaucoma. As to provide theoretical basis for Chinese medicine treatment and prevention of liver depression syndrome glaucoma and development promotion of optic nerve protection of new medicine.

青光眼是高致盲性眼病，有明显的体质特征，肝郁证是青光眼最常见的辩证分型。研究显示约30%的蛋白质编码基因由miRNA调控，提示体质特征与miRNA密切相关。在视网膜和眼部其他组织中，miRNA的表达具有组织特异性和发育阶段特异性，提示其在视网膜和眼部其他组织具有潜在的组织和细胞的功能特异性。本课题以中医藏象理论为指导，以通窍明目4号为干预手段，以大鼠视神经节细胞（RGCs）的miRNA为研究靶点，采用miRNA芯片杂交、RT-PCR检测、P-VEP和细胞超微结构检测等方法，分别从体内体外两个方向研究肝郁证青光眼体质特征的病理基础，疏肝通窍法对肝郁证青光眼的疗效与调控基因转录的相关性；通过筛查经中药干预后的RGCs中差异表达的miRNAs，预测并验证其靶基因。从整体、基因调控及细胞等多个层次研究肝郁证青光眼的发病机制。为中药防治肝郁证青光眼，开发推广视神经保护新药提供理论依据。

背景和方向： 青光眼是高致盲性眼病，有明显的肝郁体质特征。研究显示miRNA在视网膜和眼部其他组织具有潜在的组织和细胞的功能特异性。中医药在治疗青光眼性视神经萎缩方面有着独特的优势，目前迫切需要基础研究做理论支撑。本研究探讨了肝郁证慢性高眼压大鼠视网膜神经节细胞miRNAs表达特征及疏肝通窍法对其表达的影响，为中药防治肝郁证青光眼，开发推广视神经保护新药提供理论依据。.主要内容：本研究以通窍明目4号为干预手段，以大鼠视神经节细胞（RGCs）的miRNA为研究靶点，采用miRNA芯片杂交、RT-PCR检测、P-VEP和细胞超微结构检测等方法，分别从体内体外两个方向研究肝郁证青光眼体质特征的病理基础，疏肝通窍法对肝郁证青光眼的疗效与调控基因转录的相关性；通过筛查经中药干预后的RGCs中差异表达的miRNAs，预测并验证其靶基因。.重要结果: miR-184为高眼压及肝郁高眼压大鼠视网膜共同差异性表达的miRNA，miR-133b-5p、miR-1b为肝郁证慢性高眼压大鼠视网膜特异性表达的miRNA。疏肝通窍法可能是通过调控miR-133b-5p、miR-211-5p、miRNA-132-3p、-222-3p、-29b-3p和-221-3p的表达发挥视神经保护作用的，其中miR-133b-5p、miRNA-29b-3p和-221-3p是疏肝通窍法具有特异性表达的miRNA。疏肝通窍法对青光眼视神经保护作用可能是通过调控调控miRNA表达，调节其靶基因和相关下游通路来完成的。.关键数据及其科学意义：.首次通过巩膜上静脉注射高渗盐水结合慢性不可预知性应激能够成功诱导出肝郁证慢性高眼压大鼠模型，为建立慢性高眼压大鼠模型的标准化操作规范提供依据。首次采用芯片技术筛选肝郁证慢性高眼压大鼠差异性表达的miRNA，发现并验证了肝郁证慢性高眼压大鼠特异性表达的及病变后期特异性表达的miRNA。.本研究经生物信息学分析预测的基因和细胞通路靶点，为日后研究肝郁证青光眼视神经损害的发病机制及病理过程提供参考。从整体、微观及基因调控等多个层次探讨了肝郁证青光眼发生的可能机制，为中医药治疗青光眼视神经损害提供有效依据，为中医药在国际市场的推广和应用铺平道路。