【作者】 王宏伟；

【导师】 郑雅娟；

【作者基本信息】 吉林大学 ， 眼科学， 2014， 博士

【摘要】 原发性开角型青光眼（Primary Open-Angle Glaucoma，POAG）是由于眼内压升高而引起的一种不可逆的致盲眼病，其致病机制与小梁细胞形态、骨架结构及生理功能密切相关。近期相关文献报道小梁细胞骨架结构发生异常改变，能引起眼压升高，有可能诱发原发性开角型青光眼。氯离子通道家族具有调控生物体内细胞的兴奋度、跨上皮细胞内的物质转运、pH值及生物体内细胞容积等多种功能，另外还对细胞分化、细胞增生、细胞凋亡及免疫应答等生理功能有着一定的调控作用。电压门控性氯离子通道(Voltage-Gated Chloride Channel，简称ClC)是氯离子通道家族的一员，目前共发现9种ClC型氯离子通道，ClC-2是ClC型氯离子通道中的一种。本项目组已证实ClC-2存在于小梁细胞，并且能调控小梁细胞多项生理活动。本实验应用RNA干扰技术，观察ClC-2表达与小梁细胞骨架结构的关系，并探讨ClC-2对小梁细胞骨架结构影响的作用机制。方法：1）ClC-2-siRNA设计、合成及活性筛选：根据ClC-2基因mRNA序列，利用计算机相关软件设计合成ClC-2-siRNA，利用Realtime-PCR，Western Blot技术检测ClC-2-siRNA对ClC-2表达的影响。2）ClC-2对人小梁细胞骨架的影响：利用Real time-PCR，WesternBlot及免疫荧光技术检测ClC-2-siRNA对Actin，Vinculin，β-catenin表达的影响。3）ClC-2对人小梁细胞骨架结构影响的机制研究：构建人小梁细胞体外青光眼模型；利用Real time-PCR，Western Blot检测体外青光眼模型小梁细胞ClC-2的表达；利用Western Blot检测TGF-β/Smad信号转导通路的表达。结果：1）本实验引进美国ScienCell公司的正常人小梁细胞作为实验细胞株，并且应用了相应的成纤维细胞培养基，细胞单层螺旋状排列，为梭形，生长良好，成功进行了传代及冻存，为后续实验奠定了良好基础。由于目前没有特异的针对ClC-2的阻断剂,为了研究ClC-2的活性与小梁细胞骨架的关系，因此我们应用了RNAi干扰技术。根据ClC-2基因序列和RNAi的设计原则，应用siRNA设计软件，分别设计了ClC-2-siRNA1，ClC-2-siRNA2.同时本文针对ClC-2-siRNA1设计了反相siRNA(scRNA)作为对照。结果证实ClC-2-siRNA最佳的转染浓度是100pmol，最佳作用时间是转染后24h。ClC-2-siRNA1对ClC-2mRNA及蛋白表达的抑制效果优于ClC-2-siRNA2。2）小梁细胞骨架的正常结构是维持小梁细胞功能的重要基础。我们主要研究ClC-2活性与小梁细胞骨架结构是否存在某种关联，是否与原发性开角型青光眼的发病机制有关系？肌动蛋白和粘着斑蛋白是小梁细胞主要的骨架蛋白，β-链蛋白是一种黏附因子，能增加细胞之间的粘合程度。我们用免疫荧光染色技术、RT-PCR、Western Blot的方法对比了正常小梁细胞和转染后的小梁细胞骨架的变化，以及肌动蛋白、粘着斑蛋白和β-链蛋白表达变化。结果发现转染后的小梁细胞的细胞骨架受到了一定的影响，细胞骨架的肌动蛋白发生紊乱，肌动蛋白肌丝出现了扭曲交联，微丝虽然增加但失去了正常的排列。黏着斑蛋白排列发生了变化，无法沿着肌动蛋白微丝进行有序排列。同时转染后的小梁细胞的肌动蛋白、粘着斑蛋白和β-链蛋白的蛋白含量、mRNA表达较正常增加，差异有统计学意义。3）小梁细胞体外青光眼模型的细胞形态与正常小梁细胞区别不大，主要是细胞体积略大，核偏圆，排列略显不规则。我们应用RT-PCR、Western Blot的方法检测体外青光眼模型小梁细胞的ClC-2的表达较正常小梁细胞下降，差异具有统计学意义；其肌动蛋白、粘着斑蛋白和β-链蛋白的蛋白含量、mRNA表达较正常增加，差异具有统计学意义。转化生长因子（TGF-β）在POAG发病机制中起着重要的作用，为了探讨ClC-2活性对小梁细胞骨架影响的机制，我们应用RT-PCR、Western Blot方法检测了ClC-2-siRNA转染后的小梁细胞TGF-β和Smad2的表达。结果发现ClC-2-siRNA转染后的小梁细胞的TGF-β和Smad2的表达较正常小梁细胞增加，差异有统计学意义，推测TGF-β/Smad转导通路可能在ClC-2对小梁细胞骨架影响过程中发挥了重要作用。结论：1）ClC-2对人小梁细胞骨架结构具有保护作用。2）ClC-2对人小梁细胞骨架结构具有的保护作用可能与TGF-β/Smad信号转导通路有关更多还原

【Abstract】 Primary open angle glaucoma(POAG) is due to an irreversibleneurological diseases caused by elevated intraocular pressure, it is closelyrelated to the pathogenesis and trabecular cell skeletal structure,physiological function and morphology. Recent reports about trabecularcell cytoskeleton structure changes show, such as the impact of theaqueous humor （AH） permeability caused by aqueous humoraccumulation or narrowing clearance of trabecular cell leading toobstructing aqueous humor discharging，can cause increased intraocularpressure，finally maybe induce primary open angle glaucoma.Chloride channel family can regulate the excitement of organismcell, transport substance of epithelial cell，regulate pH value or theorganism cell volume.In addition chloride channel family also regulatemany physiological functions， such as the cell differentiation, cellproliferation, apoptosis and immune response et al. ClC is an abbreviationfor Voltage-Gated Chloride Channel,as a member of the family ofchloride channels. At present there have been9different types of ClCchloride channel, ClC-2is a type ClC chloride channel. The project teamhas confirmed that ClC-2exists in the trabecular meshwork cell, and canregulate many physiological activities of trabecular meshworkcell.Purpose of this experiment is observing the relationships between theexpression of ClC-2and trabecular meshwork cytoskeleton structure, andinvestigating mechanism of ClC-2on cytoskeleton structure and function of trabecular meshwork cell by RNA interference technique.Methods：1）Design and selection of ClC-2-siNRA: Briefly, a region ofClC-2-siNRA was designed according to ClC-2mRNA and computerprediction. Real time-PCR and western blotting analyses were used todetect the effects of ClC-2-siNRA on the expression of ClC-2(mRNAand protein) in different concentration and different time.2）The effects of ClC-2on the cytoskeleton in human trabecularmeshwork cell: Real time-PCR, Western Blot and immunofluorescencetechnique were used to detect the effects of ClC-2-siRNA on Actin,Vinculin, and β-catenin expression.3）The mechanism of ClC-2on the cytoskeleton in human trabecularmeshwork cell： Construct glaucoma model of human trabecularmeshwork cell in vitro；Real time-PCR and western blot were used todetect the expression of ClC-2in VGM cells；western blot were used todetect the expression of TGF-β/Smad signal pathway.Results:1）In this experiment，we introduce the normal human trabecularcells from America ScienCell as experimental cell line，and apply thecorresponding fibroblast culture medium.One-layer cells spirally arrangeand show spindle，its growth is good.The success of subculture andcryopreservation lays a good foundation for the subsequentexperiment.Because there is no specific for the blocking agent of ClC-2，we use RNA interference technology to study the relationship betweenClC-2activity and trabecular meshwork cytoskeleton.According to thedesign principle of ClC-2gene sequence and RNAi， we designClC-2-siRNA1and ClC-2-siRNA2by using the siRNA design software.At the same time，we design reverse siRNA (scRNA) forClC-2-siRNA1as control. The results confirm that the optimumconcentration of ClC-2-siRNA is100pmol，and the best time is24h aftertransfection.The inhibitory effect of ClC-2-siRNA1on the expression ofClC-2mRNA and protein is better than other siRNAs.2） The integrity of normal structure of trabecular meshworkcytoskeleton is an important foundation to maintain trabecular cellfunction.Then we study whether there is any relation between the activityof ClC-2and trabecular cytoskeletal structure，whether there is anyrelation with the pathogenesis of primary open angle glaucoma.Actin andvinculin are cytoskeletal protein of trabecular cells，and beta catenin is akind of adhesion factor，it can increase the adhesion level betweencells.We use immunofluorescence staining technique，RT-PCR，WesternBlot to contrast changes in cytoskeleton，actin，vinculin and beta cateninprotein expression between normal trabecular cells and trabecular cellsafter transfection.The results find that cytoskeleton of transfectedtrabecular cells is affected a little，cytoskeletal actin occurs disorder，thereare cross-linked actins.Although microfilaments increase，but they losethe normal arrangement.Vinculin arrangement changes，and it does notorderly arrange along the actin filaments.At the same time protein andmRNA expression of actin，vinculin and beta catenin of trabecular cellsafter transfection are higer than normal trabecular cells，the difference isstatistically significant.3）There is little difference in cellular morphology of normaltrabecular cells and trabecular cells in vitro glaucoma model，only the cellvolume of trabecular cells in vitro glaucoma model is slightly larger， nuclear is rounder，slightly irregular arrangement.We also use RT-PCR，Western Blot to detect ClC-2expression of trabecular cells in vitroglaucoma model，the result is ClC-2expression of its is less than normaltrabecular cells， the differenc is statistically significant.Protein andmRNA expression of its actin，vinculin and beta catenin are higher thannormal trabecular cells， the difference is statistically significant.Transforming growth factor (TGF-β) plays an important role in thepathogenesis of POAG，in order to investigate mechanism of ClC-2activity on trabecular cytoskeleton，we use RT-PCR，Western Blot todetect the expression of TGF-β and Smad2of trabecular cells aftertransfection.The study finds that expression of TGF-β and Smad2intrabecular meshwork cells after transfection are higher than normaltrabecular cells，the difference was statistically significant.We speculatethat the mechanism of TGF-β/Smad2pathway may be involved in theeffect of ClC-2on trabecular meshwork cytoskeleton.Conclusions：1．ClC-2could have a protective effect on the cytoskeleton in humantrabecular cell.2．The effect mechanism of ClC-2might be related with TGF-β/Smad signal pathway on the cytoskeleton in human trabecular cell.更多还原