RESUMO
BackgroundResearches demonstrated that the levels of soluble CD44 (sCD44)molecule in aqueous is significantly higher in primary open-angle glaucomous(POAG) eye than normal eye,but how the sCD44 would affect the expression of apoptosis protein in trabecular meshwork cells is below understanding.Objective The present study was to investigate the effect of sCD44 on the expression of regulatory proteins bcl-2 associated death factor bad in trabecular meshwork cells in the patients with POAG.MethodsHuman scleral tissue with trabecular meshwork were obtained from POAG patients during the surgery.The trabecular meshwork cells were primarily cultured by explant culture method and identified by immunochemistry.The third generation of cells were incubated with free-serum DMEM/F12 medium added differnt dosages of sCD44 (0,1,5,10,25,50 mg/L) for 48 hours.The expression of bad protein in cultured cells was detected using cell counting kit-8 (CCK-8) as the absorbance values at 490 nm(A,90 value),and the bad protein level in cultured cells was assayed by ELISA.ResultsThe cultured cells showed the positive response for laminin ( LM ),neuron specific enolase ( NSE ),fibronectin ( FN ) monoclonal antibodies.The CCK-8 assay showed that the A490 values of the trabecular meshwork cells in 0,1,5,10,25,50 μg/L of sCD44 groups were 0.2460±0.0019,0.1874±0.0015,0.1570±0.0016,0.1302±0.0019,0.1084±0.0018,0.0940±0.0020 respectively with a statistically significant difference among the 6 groups( F =14.922,P =0.000 ),and the A490 values in various dosages of sCD44 groups were significantly lower than the 0 μg/L sCD44 group (P=0.013,0.008,0.011,0.005,0.004).The ELISA assay showed that bad protein levels in 0,1,5,10,25,50 μg/L of sCD44 groups were ( 114.8461 ± 2.9560 ),( 137.8270 ± 2.4259 ),( 161.4194 ± 3.7381 ),( 170.9453 ± 3.2006 ),( 221.2252 ±4.3738 ),( 324.6167±4.4220) ng/L,showing a total difference among them ( F =16.610,P =0.000 ),and the bad protein levels in various dosages of sCD44 groups were significantly lower than the 0 μg/L sCD44 group( P =0.017,0.013,0.008,0.007,0.006).ConclusionssCD44 can contribute to the apoptosis of the trabecular meshwork cells in patients with POAG in certain dose range by regulating the apoptosis regulatory proteins bcl-2 associated death factor bad.
RESUMO
Background Primary open angle glaucoma (POAG) is a major blindness-causing disease,characterized by elevated intraocular pressure due to an insufficient outflow of aqueous humor. The trabecular meshwork lining the aqueous outflow pathway modulates the aqueous outflow facility. To study the biological characteristics of the trabecular meshwork cells has important significance. Objective This study was to culture the trabecular cells from primary open-angle glaucomatous eye (POAG) and study the biologic characteristics of passaged cells. Methods The deep scleral tissue with trabecular meshwork was obtained during the trabeculectomy from 8 eyes with POAG. The trabecular cells were primarily cultured and passaged in vitro. The generation 3 cells were identified by immunochemistry with the laminin (LM), fibronectin (FN) and neuron specific endolase (NSE)monoclonal antibodies. The ultrastructure was examined to observe the biological characteristics of the cells under the transmission electronic microscope. The experimental results were compared among POAG group, normal control group and blank control group. Results The primarily cultured POAG trabecular cells migrated from the edge of tissue mass about 10 days. The cells of generation 3 presented the logarithmic phase in the first 4 days and fused in the 7th day. FN,LM and NSE were positively expressed in the generated cells in POAG group and normal control group rather than blank control group. The MOD values of the generation 3 cells for FN in POAG group and normal control group were 0. 35 ± 0.06 and 0. 26 ± 0. 01, and those for LM were 0. 34 ± 0. 03 and 0. 25 ± 0. 02 respectively, showing statistically significant difference between these two groups ( FN: t = 14. 446, P＜0.001; LM: t = 9. 346, P＜0. 001 ). The microvilli, cytolysosome and phagocytic vesicle were obviously decreased in the trabcular cells of POAG group compared with normal control group under the transmission electron microscope. Conclusion The trabecular meshwork cells from POAG can be successfully cultured and passaged in vitro. It provides a cytology basis for further glaucoma research.