氯离子通道在青光眼视神经保护中的作用机制研究

Glaucoma is the first leading causes of irreversible blindness all over the world. The final common pathway of glaucomatous optic neuropathy is retinal ganglion cell(RGCs) apoptosis. Because lowing intraocular pressure is not always sufficient to halt the progression of this optic neuropathy,there has been considerable effort to develop therapeutics that prevent or delay the RGCs from undergoing degeneration. However the mechanism of RGCs apoptosis is not very clear. Recently the study on ion channel involved in the RGCs apoptosis has stepped, especially the effect of chloride channel on RGCs is focused on. Chloride channels (ClC) have been shown extensively distributing in mammal's organs, tissues and cells, which play an important role in many physiological and pathological functions including maintaining cells volume, regulating cell electrical activity，proliferation，apoptosis，migration and so on. Using RT-PCR and cellular immune dyeing technology, we first confirmed that some chloride channels including ClC-2、ClC-3、ClC-5 expressed in RGC-5 at mRNA and protein level. Researches have confirmed that activitied volume-sensitive chloride channels in low permeability state could protect neural apoptosis by reducing glutamic acid release in astroglia. Effects of chloride channel blocker in proliferation and apoptosis may relate to chloride channels' regulations to Ca2+ channels directly. By restraining chloride channel, chloride channel blocker changes activity and intracellular concentration of Ca2+ channel, which named calcium overload results in apoptosis of RGCs. Thus indicate that chloride channels play an important role in apoptosis, and also we have found that ClC-3 could protect apoptosis of RGC-5 induced by glutamic acid in the earlier experiment. This study We will through the in vitro culture retinal ganglion cells line (RGC-5), set up the pressure induced RGC-5 cell apoptosis model, observed the changes of the viability、apoptosis of RGC-5 and activity of chlorine channels pretreated with multiple pressure,different time duration, and investigate the effect and control way of volume-sensitive chloride channel ClC-2 and ClC-3 on RGCs apoptosis. In order to determine the role of chloride channel in RGCs apoptosis. Based on the above results, we'll observe the effect of chloride channel blockers,agonists on the RGCs apoptosis's occurrence and development, so as to offer the theoretical bases to glaucomatous optic neuroprotection.

青光眼是全球第一位的不可逆性致盲眼病。视网膜神经节细胞(retinal ganglion cells，RGCs)凋亡是青光眼视神经损伤的最终共同通路。由于控制眼压有时并不能完全阻止视神经的损伤，很多学者正试图寻找能阻断或延缓神经节细胞损伤的方法。但RGCs凋亡的发生机制目前尚不完全清楚，最近有关离子通道参与RGCs凋亡的研究正逐步深入，特别是氯离子通道的作用更受到重视。本研究将通过体外培养视网膜神经节细胞永生株(RGC-5)，建立压力诱导的RGC-5细胞凋亡模型，观察在多个压力、时间点下，RGC-5细胞活力、凋亡及氯离子通道ClC-2、ClC-3活性的变化，探讨分布于RGC-5上的ClC-2、ClC-3在RGC-5凋亡中的作用和调控途径，以阐明RGC-5凋亡的发生机制；并利用大鼠青光眼动物模型，观察氯离子通道阻滞剂、激动剂对RGCs凋亡发生、发展的影响，为青光眼视神经保护治疗提供理论依据。

视网膜神经节细胞(retinal ganglion cells, RGCs)的凋亡是视神经损伤、青光眼、缺血性视神经病变等眼科疾病的重要病理特征之一,也是导致视野缺损的主要原因。谷氨酸是视网膜的主要兴奋性递质,正常情况下不引起毒性。但在眼压升高等原因导致视网膜缺血缺氧时,谷氨酸会大量释放,对RGCs产生毒性作用。谷氨酸的过度表达是导致RGCs凋亡的重要原因。氯离子是生物体内含量最丰富的阴离子,通过跨膜转运和离子通道参与机体多种生物功能。电压门控性氯通道(voltage-gated chloride channnel, ClC)在哺乳动物细胞中广泛表达,ClC-2氯离子通道作为此家族中的一个亚型,是目前研究较为广泛和明确的一种氯离子通道类型,与细胞增殖、凋亡及细胞周期等多种生理功能的调节有关。关于电压门控性氯通道参与细胞凋亡过程己得到广泛证实。鉴于此,首先我们以谷氨酸诱导RGCs凋亡作为研究对象,应用cDNA转染和RNAi方法分别改变细胞内ClC-2的表达,观察细胞凋亡、细胞周期变化情况及Bcl-2及Bax蛋白表达、caspase-3、caspase-9酶活性变化,以探讨ClC-2及Bcl-2/Bax调控的线粒体凋亡通路在此过程中的作用。其次，我们探讨了压力与RGCs ClC-2 mRNA表达及细胞活力、凋亡的关系。证实：在一定的加压应激条件下，ClC-2可以一定程度维持RGCs的正常细胞活力，降低压力诱导的RGCs凋亡指数；但压力过大或者压力持续过长，ClC-2的适应性细胞保护作用将大大降低。压力应激条件下，RGCs不但有ClC-2和Hsp90蛋白的表达，而且二者以复合物的形式存在。我们使用Hsp90的特异性抑制剂格尔德霉素（GA），发现GA不能改变ClC-2 mRNA的表达，但可以通过提高ClC-2的活性、提高ClC-2对细胞内氯离子浓度的敏感性的方式对ClC-2加以调节。结论ClC-2氯通道对谷氨酸或压力诱导的RGCs凋亡具有保护作用,而Bcl-2/Bax调控的线粒体凋亡通路可能参与此过程。另外，Hsp90对氯离子通道ClC-2的电生理活性起正性调节作用。我们的实验结果提示ClC-2氯通道可能成为视神经保护的一个新靶点,为进一步研究视神经保护提供了理论基础和实验依据。