【摘要】： 原發(fā)性開角型青光眼(primary open angle glaucoma,POAG)是主要的不可逆性致盲眼病之一,其中高眼壓是青光眼性視神經(jīng)損傷的主要危險(xiǎn)因素。大量研究表明,眼壓升高的原因是房水外流阻力增高,其病理部位已鎖定小梁網(wǎng)。而小梁細(xì)胞作為小梁網(wǎng)的內(nèi)皮細(xì)胞,其形態(tài)、結(jié)構(gòu)和功能的異常將引起房水外流受阻,導(dǎo)致POAG的發(fā)生和發(fā)展[1]。 POAG的主要病理變化包括小梁細(xì)胞數(shù)量、細(xì)胞間連接、細(xì)胞外基質(zhì)的改變,以及小梁細(xì)胞的凋亡等。而有關(guān)離子通道,尤其是ClC(chloride channel)氯離子通道的研究表明,氯離子通道具有維持細(xì)胞的容積平衡、調(diào)節(jié)細(xì)胞電活動(dòng)的功能,并且在細(xì)胞增殖、遷移、凋亡等多種生理、病理過(guò)程發(fā)揮重要作用[2-3],而這些作用與POAG的主要病理改變密切相關(guān)。氯離子通道廣泛分布于哺乳動(dòng)物的組織、器官和各種細(xì)胞中,目前已克隆出從ClC-1到ClC-7及ClC-ka、ClC-kb等九種ClC氯離子通道家族成員。通過(guò)RT-PCR已證實(shí)人小梁細(xì)胞表達(dá)ClC-2、ClC-3、ClC-5等多種氯離子通道[4];David指出氯離子通道在維持小梁細(xì)胞體積、調(diào)節(jié)房水外流易度、保持小梁細(xì)胞體內(nèi)平衡等方面發(fā)揮重要作用[5]。 本研究以小梁細(xì)胞及氯離子通道家族的一個(gè)亞型ClC-3作為研究對(duì)象,探討ClC-3氯離子通道在小梁細(xì)胞中的作用。首先應(yīng)用RT-PCR及細(xì)胞免疫化學(xué)方法,檢測(cè)ClC-3氯離子通道在永生株人眼小梁細(xì)胞中的表達(dá)。然后應(yīng)用氯離子通道阻滯劑NPPB,通過(guò)MTT法、細(xì)胞周期同步化、5-FU誘導(dǎo)細(xì)胞凋亡、羅丹明123細(xì)胞線粒體膜電位(△Ψm)檢測(cè)及建立小梁細(xì)胞吞噬乳膠株模型等方法,分別研究NPPB對(duì)小梁細(xì)胞增殖、細(xì)胞周期、細(xì)胞凋亡、線粒體膜電勢(shì)及細(xì)胞吞噬功能的影響。由于氯離子通道阻滯劑NPPB對(duì)于多數(shù)氯離子通道均有阻斷作用,對(duì)于ClC-3氯離子通道只具有相對(duì)特異性,因此我們采用反義寡核苷酸技術(shù),特異性地抑制ClC-3氯離子通道蛋白在小梁細(xì)胞的表達(dá),進(jìn)而檢測(cè)ClC-3氯離子通道在小梁細(xì)胞增殖、細(xì)胞周期、細(xì)胞凋亡、線粒體膜電勢(shì)及細(xì)胞吞噬中的作用,從而為原發(fā)性開角型青光眼發(fā)病機(jī)制提供進(jìn)一步的實(shí)驗(yàn)依據(jù)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R775

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 隋海心,任罡;水分子通道蛋白的結(jié)構(gòu)與功能[J];化學(xué)進(jìn)展;2004年02期

2 張海燕;李輝;高志安;;p16、Rb、cyclinD_1蛋白在子宮內(nèi)膜腺癌中的表達(dá)及意義[J];遼寧醫(yī)學(xué)院學(xué)報(bào);2007年06期

3 張朝陽(yáng),何慶泗,劉博,王偉,馮國(guó)棟,楊高祿,胡桂青,霍景,李培;5-氟脲嘧啶誘導(dǎo)人膽囊癌細(xì)胞凋亡的研究[J];山東醫(yī)藥;2003年17期

4 張光平;背景氯離子通道研究進(jìn)展[J];生物化學(xué)與生物物理進(jìn)展;1998年04期

5 鐘寧,方奇志,金滿文,周兆年;血管內(nèi)皮細(xì)胞容量激活的氯通道[J];生命科學(xué);2000年03期

6 ;Regulation of swelling-activated chloride channels in embryonic chick heart cells[J];Cell Research;2003年01期

7 張海寧,關(guān)永源;ClC-3氯通道研究進(jìn)展[J];中國(guó)藥理學(xué)通報(bào);2003年02期

8 張海寧;丘欽英;關(guān)永源;;ClC-3氯通道對(duì)Thapsigargin觸發(fā)的Ca~(2+)運(yùn)動(dòng)的影響[J];中國(guó)藥理學(xué)通報(bào);2008年08期

9 陳飛,魏厚仁,曾艷彩,呂源淑,張櫻(;維拉帕米對(duì)牛眼小梁細(xì)胞增殖和吞噬功能的影響[J];眼科研究;2003年03期

10 侯晉;段小紅;司徒鎮(zhèn)強(qiáng);;阻斷氯離子通道對(duì)小鼠成牙本質(zhì)細(xì)胞MDPC-23生物學(xué)特性影響的研究[J];牙體牙髓牙周病學(xué)雜志;2007年06期

，

本文編號(hào)：2143116