胰岛素对正常糖和高糖浓度下牛视网膜微血管内皮细胞血管内皮生长因子表达的影响

目的 探讨胰岛素、糖浓度及其两者的联合作用对牛视网膜微血管内皮(BRE)细胞血管内皮生长因子(VEGF)表达的影响.方法 对照实验研究.采用选择性培养方法,培养BRE细胞,传代后分别在正常糖(5 mmol/L)或高糖(30 mmol/L)浓度下培养3 d,甘露醇平衡渗透压,血清饥饿12 h,给予或不给予100 nmol/L胰岛素作用24 h.实时荧光定量检测VEGF mRNA表达水平;人脐静脉血管内皮细胞增殖法、免疫荧光检测法、免疫印迹法检测VEGF蛋白表达水平.采用SPSS 12.0统计学软件对数据进行分析,胰岛素、糖浓度及其交互作用对BRE细胞的VEGF mRNA和VEGF蛋白表达水平的影响,采用2×2析因设计定量资料方差分析,以P<0.05作为差异有统计学意义.结果 胰岛素或高糖浓度可以显著提高BRE细胞的VEGFmRNA(F=5.67,9.04;均P<0.05)和VEGF蛋白(F=5.50,5.57;均P<0.05)表达水平,但胰岛素联合高糖浓度的作用较其单独作用减弱.结论 高糖浓度下可以降低胰岛素诱导的VEGF表达水平,因此,VEGF可能不是胰岛素治疗导致的糖尿病视网膜病变短期恶化的主要因素.

Effect of insulin on VEGF expression in bovine retinal microvascular endothelial cells exposed to normal or high glucose

Objective To investigate the effect of insulin or (and) high glucose on the vascular endothelial growth factor (VEGF) expression in bovine retinal microvascular endothelial cells (BREC).Methods It was a experimental study. BREC were cultured with selective culture media. Passaged cells were cultured in normal (5 mmol/L) or high glucose (30 mmol/L) medium for 3 days. Equimolar mannitol was supplemented for osmotic controls. Cells were serum-deprived overnight in Dulbeceo's modified Eagle's medium containing 0.2% w/v bovine serum albumin and then incubated in the absence or presence of 100nmol/L insuhn for 24 hrs. Cells were harvested and the expression of VEGF mRNA was analyzed by Realtime PCR. VEGF protein level was determined by HUVEC proliferation assay, immunofluorescence and western-blot. Results Insulin or (and) high glucose significantly increased VEGF mRNA ( F = 5.67,9.04 ;P <0.05) and protein (F=5.50,5.57;P<0.05) expression. However, the combined effect of insulin and high glucose is weak in contrast to insulin or high glucose alone. Conclusions High glucose attenuated insulin-induced VEGF expression. Therefore, VEGF may be not the major factor that lead to transient worsening of diabetic retinopathy after acute insulin therapy in diabetes.