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目的研究高糖对肾小球系膜细胞间隙连接蛋白(connexin43)表达和细胞间通讯功能的影响。方法分离培养大鼠肾小球系膜细胞,调整培养液葡萄糖浓度为以下3组:正常葡萄糖组(5.5mmol/L葡萄糖)、高糖组(30mmol/L葡萄糖)和渗透压对照组(5.5mmol/L葡萄糖加24.5mmol/L甘露醇),于37℃5%CO2条件下培养24、48h后,利用激光共聚焦显微镜和荧光漂白恢复(FRAP)技术检测细胞间通讯功能,并应用Northern印迹和细胞免疫化学、Western印迹方法检测connexin43mRNA和蛋白质表达,比较3组之间的差异。结果正常葡萄糖浓度培养下系膜细胞表达丰富的connexin43,细胞间通讯功能良好。高糖培养的系膜细胞细胞间通讯功能下降,荧光淬灭后的恢复比例和速度显著低于正常糖组(P<0.05)。同时高糖环境下培养的系膜细胞connexin.43mRNA和蛋白质表达均较正常糖组显著下降(P<0.05)。渗透压对照组与正常糖组之间差异无统计学意义(P>0.05)。结论高糖可抑制connexin43的基因和蛋白质表达及细胞间通讯功能,可能是糖尿病肾病系膜细胞表型和功能异常的重要原因之一。
Objective To investigate the effect of high glucose on the expression of connexin43 and the function of intercellular communication in glomerular mesangial cells. Methods Rat glomerular mesangial cells were isolated and cultured. The glucose concentration of the culture medium was adjusted to the following three groups: normal glucose group (5.5mmol / L glucose), high glucose group (30mmol / L glucose) and osmotic pressure control group / L glucose plus 24.5mmol / L mannitol) were cultured in 37 ℃ under 5% CO2 for 24,48 hours. The function of intercellular communication was detected by laser scanning confocal microscopy and fluorescence recovery (FRAP) Immunocytochemistry and Western blotting were used to detect the expression of connexin43 mRNA and protein. The differences between the three groups were compared. Results Under normal glucose concentration, mesangial cells expressed abundant connexin43 with good intercellular communication. Mesangial cells cultured in high glucose decreased the cell-to-cell communication function, and the recovery ratio and speed after fluorescence quenching were significantly lower than those in normal glucose group (P <0.05). Meanwhile, the expression of connexin.43mRNA and protein in mesangial cells cultured in high glucose were significantly decreased compared with normal glucose group (P <0.05). There was no significant difference between osmotic pressure control group and normal glucose group (P> 0.05). Conclusion High glucose can inhibit the gene and protein expression of connexin43 and the intercellular communication function, which may be one of the important reasons for the phenotypic and functional abnormalities of mesangial cells in diabetic nephropathy.