| 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. |
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