高浓度葡萄糖体外对大鼠视网膜 Müller细胞活性的影响及其机制探讨

摘要

目的：观察高浓度葡萄糖对体外培养大鼠视网膜Müller细胞活性的影响，探讨其作用机制。方法体外传代培养大鼠视网膜Müller细胞，随机分为N、G15、G25、G35组，分别给予DMEM培养基及含15、25、35 mmol／L D-葡萄糖的DMEM培养基培养5 d。采用MTT法检测各组细胞光密度值（ OD值，代表细胞活性），采用细胞免疫化学染色、免疫荧光（ FITC标记）染色及酶联免疫吸附试验检测低氧诱导因子1α（ HIF-1α）和促红细胞生成素（ EPO）蛋白。结果 N、G15、G25、G35组细胞OD值分别为0．83±0．02、0．55±0．02、0．45±0．02、0．33±0．02，组间比较，P均＜0．01。 N组未见HIF-1α、EPO阳性细胞，G15、G25、G35组可见黄染的HIF-1α阳性细胞及呈绿色荧光的EPO阳性细胞。 N、G15、G25、G35组HIF-1α蛋白OD值分别为0．02±0．01、0．58±0．02、0．75±0．02、0．92±0．02，组间比较，P均＜0．01；EPO蛋白OD值分别为0．03±0．01、0．48±0．02、0．33±0．02、0．25±0．02，组间比较，P均＜0．01。结论高浓度葡萄糖可降低体外培养大鼠视网膜Müller细胞活性，促进细胞HIF-1α、EPO蛋白表达可能是其作用机制之一。%Objective To observe the effect of high glucose on the cell viability of rat retinal Müller cells cultured in vitro and to investigate the mechanism.Methods Rats′retinal Müller cells of serial subcultivation were divided into N, G15 , G25 , and G35 groups which were cultured by DMEM medium or DMEM medium containing 15 mmol/L, 25 mmol/L and 35 mmol/L glucose for five days, respectively.MTT assay was applied to detect the optical density ( OD value which represents the cell viability) in all groups.The expression of hypoxia-inducible factor-1α( HIF-1α) and erythropoietin ( EPO) was detected by immunochemistry, immunofluorescence and enzyme linked immunosorbent assay.Results The OD values of the N, G15 , G25 and G35 groups were respectively 0.83 ±0.02, 0.55 ±0.02, 0.45 ±0.02 and 0.33 ±0.02, and significant difference was found among these four groups ( all P<0.01) .There were no positive cells of HIF-1αand EPO in the N group.Cells presented yellow positive expression of HIF-1αby immunochemistry and green fluorescence posi-tive expression of EPO by immunofluorescence in the G15 , G25 and G35 groups.The OD values of HIF-1αprotein were re-spectively 0.02 ±0.01, 0.58 ±0.02, 0.75 ±0.02 and 0.92 ±0.02 in the N, G15 , G25 and G35 groups, and significant difference was found among these four groups (all P<0.01).The OD values of EPO in the N, G15, G25 and G35 groups were respectively 0.03 ±0.01, 0.48 ±0.02, 0.33 ±0.02 and 0.25 ±0.02, and significant difference was found among these four groups (all P<0.01).Conclusion High glucose may decrease the cell viability of rat retinal Müller cells cul-tured in vitro and increase the expression of HIF-1αand EPO which may be one of its mechanisms.