高糖和胰岛素对肾小球系膜细胞葡萄糖转运蛋白4mRNA表达及细胞骨架纤维状肌动蛋白的影响

Effects of high glucose and insulin on the expression of GluT4mRNA and cytoskeleton protein F-actin of glomerular mesangial cells

目的探讨高糖和胰岛素对肾小球系膜细胞(GMC)葡萄糖转运蛋白4(GluT4)mRNA表达及细胞骨架纤维状肌动蛋白(F-actin)的影响,进一步研究糖尿病肾病发生发展中GluT4及其下游分子F-actin的重要作用。方法将培养的鼠1097系膜细胞分为8组:正常对照组,生理浓度胰岛素组(10-9mol/L),低浓度胰岛素组(10-8mol/L),高浓度胰岛素组(10-6mol/L),高糖组(30mmol/L),甘露醇组,高糖加高浓度胰岛素组,高糖加生理浓度胰岛素组。采用RT-PCR法检测GluT4mRNA含量,rhodamine-phalloidin染色,激光共聚焦显微镜观察F-actin形态并测定荧光强度。结果(1)正常系膜细胞可检测到GluT4mRNA。(2)高糖组GluT4mRNA表达为对照组的58.7%(P<0.05);10-8mol/L胰岛素组、10-6mol/L胰岛素组分别为对照组的230.2%和297.2%(P<0.01);高糖加10-6mol/L胰岛素组,高糖加10-9mol/L胰岛素组分别为高糖组的170.6%和140.3%(P<0.05)。(3)高糖组F-actin荧光强度为对照组的44.5%;10-8mmol/L胰岛素组、10-6mol/L胰岛素组分别为对照组的122.4%(P<0.05)和129.6%(P<0.01);高糖加10-6mol/L胰岛素组为高糖组的183.8%(P<0.05)。(4)GluT4mRNA表达与F-actin荧光强度呈正相关(r=0.786,P<0.05)。结论(1)正常系膜细胞有GluT4mRNA表达。(2)高糖可抑制GluT4mRNA表达及促进F-actin?

Objective To investigate the effects of high glucose and insulin on the expression of GluT4mRNA and cytoskeleton protein F actin of glomerular mesangial cells(GMCs) in order to explore the function of GluT4 and F actin in the genesis and development of diabetic nephropathy (DN). Methods Cultured 1097 rat GMCs were divided into 8 groups: control,10-9mol/L insulin,10-8mol/L insulin,10-6mol/L insulin,high glucose,mannitol,high glucose plus 10-6mol/L insulin,high glucose plus 10-9mol/L insulin. Expression of GluT4 mRNA was measured by RT PCR method, F actin was stained by rhodamine pholloidin and the fluorescent intensity was calculated by image analysis system. Results (1)GluT4mRNA could be detected in normal GMC.(2)High glucose could decrease the expression of GluT4mRNA to 58.7%of the control(P< 0 05);10-8mol/L insulin,10-6mol/L insulin could increase to 230 2%, 297 2%of the control(P< 0 01) in a concentration dependent manner; high glucose plus 10-6mol/L insulin, high glucose plus 10-9mol/L insulin could increase to 170 6%,140 3%(P< 0 05) compared with simple high glucose group. (3)High glucose could decrease the fluorescent intensity of F actin to 44 5%(P< 0 01); 10-8mol/L insulin,10-6mol/L insulin could increase to 122 4%(P< 0 05), 129 6%(P< 0 01) in a concentration dependent manner; high glucose plus 10-9mol/L insulin could increase to 183 8%(P< 0 05) compared with simple high glucose group. (4)The Spearman correlation coefficient between GluT4mRNA and F actin was 0 786(P< 0 05). Conclusions (1)Normal GMCs can express GluT4 mRNA.(2)High glucose can inhibit the expression of GluT4mRNA and facilitate the depolymerization of F actin.(3)Insulin can partly reverse the effects caused by high glucose above.(4)The correlation between the expression of GluT4mRNA and the fluorescent intensity of F actin is positive.(5)GluT4 and F actin are important factors in the development of DN.