The Study Of Chloride Channel ClC-2on Trabecular Meshwork Cytoskeleton

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.