高压力通过小梁细胞骨架对房水外流系统的作用及机制研究

Glaucoma is the leading cause of irreversible blindness, high intraocular pressure (IOP) is the major risk factor. Trabecular meshwork cells in aqueous humor outflow system continuously detect and respond to mechanical forces and adapt their physiology to maintain proper cellular function. Cytoskeleton is the main structure for its function. How do trabecular meshwork cells detect high pressure and their functional changes under high pressure remains unknown. Relevant studies have confirmed that reduction of F-actin cytoskeleton decreased IOP. Our previous study showed that high pressure affected the distribution and expression of F-actin cytoskeleton in trabecular meshwork cells. In addition, high pressure reduced the expression of ZO-1, which is closely related with cytoskeleton. Therefore, we speculate that high pressure can affect the aqueous humor outflow by cytoskeleton and the functional changes of trabecular meshwork cells. In order to test our hypothesis, the following experiments will be done. 1. In vitro: immortalized normal and glaucomatous trabecular meshwork cells will be cultured in high pressure culture system to simulate the effect of acute and chronic high pressure. Changes of cytoskeleton components will be detected by Western blot and Real-Time PCR. The effect of high pressure on cellular function will be detected as well. Then shRNA will be used to investigate the role of mechanical sensitive Pannexin channel in the mechanism of high pressure on cytoskeleton of trabecular meshwork cells. 2. In vivo: the acute and chronic high IOP mice models will be used to investigate the corresponding changes in vivo, including trabecular meshwork cells, cytoskeleton and aqueous humor outflow resistance. In summary, the aim of the study is to elucidate the molecular biological effect of high pressure on cytoskeleton in trabecular meshwork cells, the regulation of aqueous humor outflow and their mechanism, which is expected to lay experimental basis for exploring the role of high IOP on pathogenesis of glaucoma.

青光眼是第一位的不可逆性致盲眼病，高眼压是最主要的危险因素。房水外流系统中，小梁细胞时刻感受力学变化以维持正常功能，细胞骨架起关键作用。但其如何感受高压力并影响细胞功能机制不详。研究表明，破坏细胞骨架F-actin可降眼压。我们前期研究证实，高压力可破坏小梁细胞骨架F-actin分布及表达，也影响与房水外流相关的紧密连接蛋白ZO-1表达。因此，我们推测高压力可能通过改变小梁细胞骨架及功能调控房水外流。为验证此假说，本课题拟行1.体外实验：利用正常及青光眼小梁细胞高压力模型，检测细胞骨架成分及细胞功能改变。利用shRNA基因沉默技术，探索与细胞骨架密切相关的Pannexin 1 机械敏感通路在高压力作用的机制。2.体内实验：利用急、慢性小鼠高眼压模型，检测小梁细胞、骨架蛋白、房水外流变化。以期阐明高压力通过小梁细胞骨架对房水外流系统的作用及机制，为探索高眼压在青光眼发病机制中的作用奠定基础。

青光眼是世界首位的不可逆性致盲眼病，高眼压是最主要的危险因素。小梁网病理学异常所产生的房水外流阻力增加，是导致高眼压的重要原因，其中小梁细胞起关键作用。小梁细胞骨架及细胞内各种信号通路，小梁细胞的数量，及其所具有的收缩、分泌、吞噬等功能，对于维持小梁网的正常功能至关重要。本课题利用高剪切力细胞模型证实，青光眼患者小梁细胞系（GTM3）对高剪切力的作用比正常人小梁细胞系（iHTM）更为敏感；分别对iHTM及GTM3进行转录组学及蛋白组学检测，获得可能与青光眼小梁网的病理学发病机制相关的细胞骨架成分（微丝、微管、中间丝）的差异、与小梁细胞收缩、吞噬及分泌功能相关的重要因子在基因及蛋白水平的表达差异，并用多种手段进行生物学验证；分别进行蛋白质-蛋白质相互作用（PPI）、GO功能及KEGG信号通路分析，获得多种与青光眼小梁细胞发病机制相关的信号通路。选择与小梁细胞的力学感受器、细胞凋亡及细胞吞噬功能密切相关的几条通路展开进一步研究：机械力敏感通道Pannexin 1蛋白及其下游的NLRP3、Caspase-1及IL-1β信号通路；CD9及其下游的整合素α4 （ITGA4）、PI3K和Akt信号通路，CD9信号在GTM3中表达降低，过表达CD9可减少小梁细胞的凋亡；细胞吞噬相关的低密度脂蛋白受体（LDLR）相关研究，LDLR在GTM3中表达降低，他汀类降脂药Lovastatin可上调GTM3中LDLR的表达，并改善小梁细胞的吞噬功能。利用正常及转基因小鼠青光眼模型，证实应用破坏细胞骨架的药物Rho激酶抑制剂（ROCKi）后，小鼠小梁网途径的房水外流阻力降低，眼压降低；此外还发现ROCKi具有促进小梁细胞增殖的作用。目前已基本完成了本项目，通过本研究为探索小梁细胞骨架及细胞功能等病理学异常在青光眼发病机制中的作用奠定了实验基础，为探寻针对小梁细胞的抗青光眼药物提供了新的治疗靶点。