【作者】 梁巍；

【导师】 郑雅娟；

【作者基本信息】 吉林大学 ， 眼科学， 2010， 博士

【摘要】 原发性开角型青光眼(primary open angle glaucoma,POAG)是一种慢性进行性视神经病变,病理性高眼压伴有典型的视神经凹陷、萎缩及视野缺损,房角是开放的[1]。POAG患者眼压升高,主要是由于房水排出的阻力增加,而产生阻力的部位主要位于小梁网。小梁细胞作为小梁网内皮细胞,其数量、结构及功能的异常,必然会引起眼压升高,导致POAG的发生、发展[2]。因此,我们致力于通过研究小梁细胞数量以及功能变化的调节因素,来揭示POAG的发病机制。氯离子通道是一种跨膜蛋白,通过细胞膜内外存在的跨膜浓度梯度,使氯离子参与细胞的体积、增殖、PH、静息电位、兴奋的调节等各种生理过程[3]。ClC型氯离子通道即电压门控氯离子通道,是目前研究最多的一类氯离子通道。ClC-2作为ClC型氯离子通道的一个亚型,具有分布广泛的特点,目前已证实氯离子通道ClC-2在脑、小肠、结肠、胃、肾、心脏等组织上均有表达[4]。有研究显示:胶质瘤细胞上ClC-2的表达上调,且ClC-2基因表达受抑制后,细胞增殖能力降低[5];使用ClC-2的特异性抑制剂Cd+可以抑制神经胶质瘤迁移[6];特发性全面性癫痫家系中发现了CLCN2的3种不同杂合子突变[7];ClC-2基因敲除小鼠小脑白质空泡变性严重[8];ClC-2缺陷使视网膜色素上皮细胞跨细胞膜运输障碍[9]。由此可见,氯离子通道ClC-2不仅在多种疾病的发生、发展中起重要的作用,而且其功能在不同组织、细胞上也并不完全相同。细胞凋亡(Apoptosis)是指有核细胞在一定条件下,通过启动其自身内部机制而发生的一种细胞自然死亡过程。已有实验证实:糖皮质激素诱导的小梁细胞凋亡是激素性青光眼发病的重要机制[10];TGF-β导致的小梁细胞凋亡增加是其促进POAG发生、发展的重要因素[11];而氯离子通道阻滞剂5-硝基-2-(3-苯丙胺)苯甲酸( 5-nitro-2-(3-phenyl-p ropylamino)-benzoic acid,NPPB)可以促进心肌细胞凋亡,增加缺血诱导的心肌梗死面积[12];使用氯离子通道阻滞剂NPPB可以诱导皮质神经元凋亡[13]。由此可见,不仅小梁细胞凋亡的发生,会对POAG的发生、发展产生重要影响,而且作为ClC型氯离子通道的一种亚型,氯离子通道ClC-2极有可能通过参与对小梁细胞凋亡的调节,进而在POAG的发生、发展中发挥重要作用。因此本实验拟通过研究氯离子通道ClC-2对人眼小梁细胞活力、凋亡的影响,及ClC-2能发挥相应作用的调节因素,来论证ClC-2与POAG之间的关系。更多还原

【Abstract】 Primary open angel glaucoma(POAG) is characterized by elevated intraocular pressure associated with optic disk cupping and visual field loss,but without the closer of anterior chamber angle.As endothelial cells of trabecular meshwork, trabecular meshwork cells (HTC) normally induce POAG if their structure、quantity and function were changed. As a kind of plasma membrane protein,ClC-2 play a key role in regulating processes of cell volume、prolif- eration、PH、ionic homeostasis and electrical excitability.For these reasons,the study about the relationship between ClC-2 and HTC will be helpful to better understand the occurrence and development of POAG.In the first part, the trabecular meshwork tissues from human eyes were primarily cultured. The morphologic features and growth characteristics of cultured cells were observed and detected by microscope and immunoc- ytochemical staining. Then we used RT-PCR and Western-Blot to determine ClC-2 mRNA and protein expression of cultured HTC in vitro.Next we constructed a pair of ClC-2 gene-specific small interfering RNA(siRNA) and its expression vector, which was transfected into cultured HTC with Lipofectamine. Trypan Blue Staining and TUNEL were applied to detect the changes of viability and apoptosis on HTC after interfered by ClC-2 siRNA.The results showed that our cultured HTC expressed the ClC-2 indeed. The interference ClC-2’s expression on HTC was able to suppress the cell’s viability,and induce HTC’s apoptosis. The abnormal increase of intraocular pressure(IOP) was the major risk factor of glaucoma to cause visual impairment.So in chapter two,we observed the relationship between pressure and mRNA expression of ClC-2、and also with the apoptosis of HTC.we next going to observed the changes of the viability、apoptosis and ClC-2 expression of HTC pretreated with different pressures by using vitality、TUNEL assay and RT-PCR. The following step was to repeat above procedures on HTC which was transfected with ClC-2 siRNA. From the results we got, we concluded that ClC-2 was able to protect the pressure-induced apoptosis in HTC partially, but the effect was diminished when the pressure was too high and the exposure time was long enough.However, what factors regulated the changes of pressure pre-treated HTC ClC-2’s effect is another issue.As we known, Hsp90 is a major factor in the procedure of adaptive cytoprotection.So we wanted to confirm if there were some connections between ClC-2 and Hsp90. In chapter three, we used Western-Blot to detect the expression of ClC-2 and Hsp90 on HTC,which was pre-treated with pressure.And then coimmunoprecipitation was used to prove that ClC-2 can interact with Hsp90. If our hypothesis is proved, the next step we were going to use GA which is the specific inhibitor of Hsp90 to treat the HTC,and then detect ClC-2 mRNA expression.The result showed that the mRNA expression of ClC-2 failed to be influenced by GA. Because the interaction between ClC-2 and Hsp90 had been proved preliminarily , whole cell patch-clamp was performed to record the currents change of GA treated and non-GA treated HTC. The result demonstrated that Hsp90 was able to effectively boost the activity of ClC-2 and enhanced channel sensitivity to [Cl-]i.In summary, we conclude that (1) HTC had the mRNA and protein expression of ClC-2;(2)ClC-2 was able to regulate viability and apoptosis of HTC;(3)ClC-2 had the effect of adaptive cytoprotection to pressure pre-treated HTC’s vitality and apoptosis; (4)Hsp90 was unable to up-regulate the ClC-2 expression in cells, which were pretreated with pressure,but was capable of enhancing the activity of ClC-2 under the pressure condition. All my experiments provide a new way to prevent and treat POAG.更多还原