Effect Of Soluble CD44 Molecules On The Apoptosis Of Cultured Trabeculat Meshwork Cells From Primaty Open Angle Glaucoma And The Investigate Of Its Apoptosis-signaling Patyway

Purpose To investigate the effect of different concentrations of soluble CD44 molecule (sCD44) in trabecular meshwork cells from primary open angle glaucoma (POAG) on the apoptosis of trabecular meshwork cells and on the expression of regulatory proteins Bcl-2 associated death factor Bad,study the relationship between sCD44 and POAG, and further investigate the pathogenesis of POAG.Methods Human trabecular meshwork cells were primarily cultured and subcultured,then identified by immunohistochemistry. to observe its biological characteristics under electronic microscope.The 3rd passage cells were incubated with different dosages of sCD44 (1,5,10,25,50ng/ml) final concentration,diluted by DMEM/F12 without serum for 24 hours,using CCK-8,fluorescence microscopy,flow cytometry method and ELISA to study the effect of sCD44 on the apoptosis of Cultured trabecular meshwork cells and on the expression of regulatory proteins Bcl-2 associated death factor Bad,Rusults 1,The primary cells in cultured were observed from the edge of the cultured tissues after 7 days. The cultured cells were cell morphology varied, fusiform, round, oval and so on under the inverted microscope.The Electron microscopy showed that the cells were round or oval, more surface microvilli and the connections between the cells were mainly gap junctions, the cytoplasm contained secondary lysosomes, rough endoplasmic reticulum, mitochondria; 2.The fibronectin(FN),laminin(LN) and NSE (NSE) were positive by Immunocytochemistry Detection,but negative controls were not color;3.With increasing concentration of sCD44,the inhibition on POAG trabecular meshwork cells were enhanced,by cell counting kit-8(CCK-8),and the experimental group compared with the control group and within experimental group was statistically significan(P <0.05 );4.By fluorescence microscopy showed that the early and late apoptotic cells were increased with the increasing final concentration of sCD44;5.The experimental group′s apoptosis rate of the different dosages of sCD44 (1,5,10,25,50ng/ml) final concentration were:(10.7283±0.0223),(17.3267±0.0250),(21.1483±0.0248),(25.1267±0.0281),(29.9900±0.0335),,by flow cytometry method,which were higher than the control group(2.5550±0.0187).The experimental group compared with the control group and within experimental group the difference was statistically significan(P <0.05) ; 6.The experimental group′s Bad protein concentration of the different dosages of sCD44 (1,5,10,25,50ng/ml) final concentration were:(89.9392±2.5995)pg/ml,(109.7353±3.9992)pg/ml,(120.1332±3.3993)pg/ml,(160.1252±5.3989)pg/ml,(220.5131±4.7990)pg/ml by ELISA assay,which were higher than the control group(71.1430±0.3999) pg/ml. The experimental group compared with the control group and within experimental group the difference was statistically significan(P <0.05).Conclusion 1.The trabecular meshwork cells from POAG can be successfully cultured in vitro.2. sCD44 could inhibit the proliferation of trabecular meshwork cells in patients with POAG,cause apoptosis, and showed a dose dependent manner;3. sCD44 could up-regulation the apoptosis regulatory proteins Bcl-2 associated death factor Bad,and had an effect on the apoptosis of trabecular meshwork cells through the endocardial line plastochondria way.