ClC-3氯离子通道在小梁细胞中作用的实验研究

发布时间：2018-07-25 07:43

【摘要】： 原发性开角型青光眼(primary open angle glaucoma,POAG)是主要的不可逆性致盲眼病之一,其中高眼压是青光眼性视神经损伤的主要危险因素。大量研究表明,眼压升高的原因是房水外流阻力增高,其病理部位已锁定小梁网。而小梁细胞作为小梁网的内皮细胞,其形态、结构和功能的异常将引起房水外流受阻,导致POAG的发生和发展[1]。 POAG的主要病理变化包括小梁细胞数量、细胞间连接、细胞外基质的改变,以及小梁细胞的凋亡等。而有关离子通道,尤其是ClC(chloride channel)氯离子通道的研究表明,氯离子通道具有维持细胞的容积平衡、调节细胞电活动的功能,并且在细胞增殖、迁移、凋亡等多种生理、病理过程发挥重要作用[2-3],而这些作用与POAG的主要病理改变密切相关。氯离子通道广泛分布于哺乳动物的组织、器官和各种细胞中,目前已克隆出从ClC-1到ClC-7及ClC-ka、ClC-kb等九种ClC氯离子通道家族成员。通过RT-PCR已证实人小梁细胞表达ClC-2、ClC-3、ClC-5等多种氯离子通道[4];David指出氯离子通道在维持小梁细胞体积、调节房水外流易度、保持小梁细胞体内平衡等方面发挥重要作用[5]。本研究以小梁细胞及氯离子通道家族的一个亚型ClC-3作为研究对象,探讨ClC-3氯离子通道在小梁细胞中的作用。首先应用RT-PCR及细胞免疫化学方法,检测ClC-3氯离子通道在永生株人眼小梁细胞中的表达。然后应用氯离子通道阻滞剂NPPB,通过MTT法、细胞周期同步化、5-FU诱导细胞凋亡、罗丹明123细胞线粒体膜电位(△Ψm)检测及建立小梁细胞吞噬乳胶株模型等方法,分别研究NPPB对小梁细胞增殖、细胞周期、细胞凋亡、线粒体膜电势及细胞吞噬功能的影响。由于氯离子通道阻滞剂NPPB对于多数氯离子通道均有阻断作用,对于ClC-3氯离子通道只具有相对特异性,因此我们采用反义寡核苷酸技术,特异性地抑制ClC-3氯离子通道蛋白在小梁细胞的表达,进而检测ClC-3氯离子通道在小梁细胞增殖、细胞周期、细胞凋亡、线粒体膜电势及细胞吞噬中的作用,从而为原发性开角型青光眼发病机制提供进一步的实验依据。
[Abstract]:Primary open-angle glaucoma (primary open angle glaucoma Poag) is one of the major irreversible blindness, in which high intraocular pressure is the main risk factor for glaucoma optic nerve injury. A large number of studies have shown that the increase of intraocular pressure is due to the increased resistance of aqueous humor outflow, and its pathological location has been locked in trabecular meshwork. As the endothelial cells of trabecular meshwork, the abnormal morphology, structure and function of trabecular meshwork cells will result in the obstruction of aqueous humor outflow, leading to the occurrence and development of POAG [1] .The main pathological changes of POAG include the number of trabecular cells, intercellular junctions. Changes of extracellular matrix and apoptosis of trabecular cells. Studies on ion channels, especially ClC (chloride channel) chloride channels, have shown that chloride channels can maintain cell volume balance, regulate cell electrical activity, and have many physiological functions, such as cell proliferation, migration, apoptosis and so on. Pathological process plays an important role [2-3], and these roles are closely related to the main pathological changes of POAG. Chloride ion channels are widely distributed in mammalian tissues, organs and various cells. At present, nine ClC chloride channel family members have been cloned from ClC-1 to ClC-7 and ClC-KaClC-kb. RT-PCR has confirmed that human trabecular meshwork cells express ClC-2ClC-3ClC-5 and other chloride channels [4] David points out that chloride channels play an important role in maintaining trabecular meshwork cell volume, regulating aqueous humor efflux, and maintaining trabecular cell balance in vivo [5]. In this study, the role of ClC-3 chloride channel in trabecular meshwork cells and a ClC-3 subtype of chloride channel family was studied. The expression of ClC-3 chloride channel in immortalized human trabecular meshwork cells was detected by RT-PCR and immunocytochemistry. Then the cell apoptosis was induced by MTT, a chloride channel blocker, and the mitochondrial membrane potential (蠄 m) of Rhodamine 123 cells was detected and the model of trabecular meshwork cell phagocytosis was established. The effects of NPPB on the proliferation, cell cycle, apoptosis, mitochondrial membrane potential and phagocytosis of trabecular meshwork cells were studied. Because the chloride channel blocker NPPB has blocking effect on most chloride channels and relatively specific for ClC-3 chloride channel, we use antisense oligonucleotide technology. To specifically inhibit the expression of ClC-3 chloride channel protein in trabecular meshwork cells, and then detect the role of ClC-3 chloride channel in the proliferation, cell cycle, apoptosis, mitochondrial membrane potential and phagocytosis of trabecular meshwork cells. Therefore, it provides further experimental evidence for the pathogenesis of primary open angle glaucoma.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R775

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