| Objectives:Diabetic retinopathy(DR),especaily proliferative diabetic retinopathy(PDR) has become the first cause of blindness in diabetic patients.Angiogenesis is the symbolic histopathological chang.Hyperglycemia and hyperinsulinemia may take part in the pathyogenesis of retina angiogenesis,so rhesus retinal endothelia cells(RF/6A)were used:1.To explore the effect of a high concentration of glucose(hereinafter called "HG") and a high concentration of insulin(hereinafter called "HI") RF/6A proliferation,migration and lumen formation.2.To investigate the synergistic effect of HG and HI on RF/6A angiogenesis.3.To elucidate the mechanism by which HG and HI affect RF/6A angiogenesis.Methods:1.Incubated RF/6A cells,after loud out the effect of glucose and insulin with different concentrations on cells using mannitol as control,divided them into the following groups:control group,glucose groups ranging 5-25 mmol/L,insulin groups ranging 1-100nmol/L supplemented with mannitol as appropriate to the osmotic pressure.2.To test the effect of each intervention factor on RF/6Aproliferation,migration and lumen formaton respectively.3.Using RT-PCR to determine the effect of each intervention factor on RF/6A's expression of VEGF mRNA,VEGFR1 mRNA,VEGFR2 mRNA using the expression of GAPDH mRNA as control.4.Using Western Blot to explore the expression of RF/6A VEGF,p-VEGFR2,P-AKT using the expression ofβ-actin as control.Results:1.Glucose dose-dependentely increased RF/6A proliferation.When glucose ranged 0-62.5mmol/L,Glucose increased RF/6A proliferation by 6.1%-15.12% (P＜0.05);There were no significant differences between each control group with the same concentration of mannitol(P＞0.05);When glucose increased 125 or 250 mmol/L,HG inhibited RF/6A proliferation by 11.87%and 16.06%respectively while control group was 12.51%and 16.87%respectively(P＜0.01).When insulin ranged 0-1000 nmol/L,dose-dependentely increased RF/6A proliferation by38.60-117.27%(P＜0.01).2.Both glucose and insulin promoted RF/6A migration by way of dose-dependent by7.52-39.24%and 51.15-148.82%(P＜0.05) respectively.3.HG and insulin promoted RF/6A tube formation by 59.1%,103-242.79%(P＜0.05).4.HG and insulin promoted RF/6A expression of VEGF mRNA,VEGFR2 mRNA and the expression ofVEGF,P-VEGFR2,P-AKT(P＜0.05).5.HG +HI could significantly strengthed HG's promotive effect on RF/6A's proliferation,migration and lumen formation(it was incresead by25.57%,51.60%,97.46%respectively,P＜0.01);while HG could partly elimited HI's promotive effect on RF/6A's proliferation,migration and lumen formation(it was reduced by 10.46%,15.17%,9.08%respectively,P＜0.01);6.HG +HI could significantly strengthed HG's promotive effect on RF/6A expression of VEGF mRNA,VEGFR2 mRNA(it was incresead by24%,25.49%,P＜0.01)and VEGF,P-VEGFR2,,P-AKT(it was incresead by 19.23%,36.6%,66.67%,P＜0.01); while HG could partly elimited HI's promotive effect on RF/6A's expression of VEGF mRNA,and VEGF(it was reduced by 8.82%,11%,P＜0.05).7.There was no difference in the expression of VEGFR1 mRNA among the intervented groups above(P＞0.05).Conclusions:1.HG and insulin promoted RF/6A proliferation,migration and lumen formation2.HG and insulin promoted RF/6Aexpression of VEGF and VEGFR2,might via intracellular signal transduction of PI3K/AKT pathway to induce angiogenesis3.HG+HI could significantly strengthed HG's promotive effect on RF/6A's angiogenesis.4.VEGFR1 might not take part in RF/6A angiogenesis mediated by both HG and HI. |
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