丙丁酚对高糖环境下人视网膜血管内皮细胞增殖及VEGF表达的影响

Effects of probucol on proliferation and expression of VEGF of human retinal capillary endothelial cells in high glucose environment

目的研究不同浓度的丙丁酚(普罗布考,Probucol)对体外培养的高糖环境下人视网膜微血管内皮细胞(human retinal capillary endothelial cells,HRCEC)增殖及血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)表达的影响。方法 体外培养从角膜移植术后新鲜人眼球提取的HRCEC。取生长良好的第3-4代细胞用于实验,实验分为低糖对照组、高糖对照组、高糖+不同浓度丙丁酚(5μmol.L-1、10μmol.L-1、20μmol.L-1、40μmol.L-1、80μmol.L-1)组,用噻唑蓝比色法(MTT)检测24h不同浓度的丙丁酚对其增殖的抑制作用。免疫细胞化学法检测低糖对照组、高糖对照组和不同浓度丙丁酚(5μmol.L-1、40μmol.L-1、80μmol.L-1)组HRCEC中VEGF的表达。结果 MTT比色法结果显示:不同浓度的丙丁酚作用于原代培养HRCEC24h,细胞增生抑制率分别为8.40%、20.84%、34.90%、45.32%、55.38%。一定质量浓度范围内丙丁酚可抑制高糖环境下HRCEC的增殖(P<0.05),24h内呈质量浓度依赖性。低糖对照组与高糖对照组相比差异无统计学意义(P=0.067>0.05)。低糖对照组、高糖对照组与丙丁酚浓度分别为5μmol.L-1、40μmol.L-1、80μmol.L-1作用于HRCEC24h,免疫细胞化学背景灰度值与阳性灰度值之差分别为28.75±3.77、71.93±8.37、54.67±4.33、41.43±3.45、29.18±3.37。与高糖对照组相比,加药组作用24h VEGF表达明显下降,差异均有统计学意义(均为P<0.05),且呈质量浓度依赖性。与低糖对照组相比,高糖对照组VEGF的表达明显增加(P<0.05)。结论 丙丁酚对体外培养的高糖环境下HRCEC的增殖及高糖引起VEGF表达的上调具有抑制作用。

Objective To investigate the effects of probucol with different concentrations on the proliferation and expression of intercellular vascular epithelial growth factor（VEGF）of human retinal capillary endothelial cell（HRCEC） in high glucose environment cultured in vitro.Methods HRCEC were extracted from fresher eyeball after corneal transplantation and cultured in vitro,which being at the 3rd to 4th generation and good growth were taken in experiment.The experimental subjects were divided into low glucose control group,high glucose control group and high glucose ＋ different concentrations（5 μmol·L-1,10 μmol·L-1,20 μmol·L-1,40 μmol·L-1,80 μmol·L-1） probucol groups.The effect of probucol on the proliferation of HRCEC for 24 hours was measured by MTT assay.Immunocytochemistry process detected the expression of VEGF in HRCEC in low glucose control group,high glucose control group and high glucose＋different concentrations（5 μmol·L-1,40 μmol·L-1,80 μmol·L-1） probucol groups.Results MTT assay indicated the inhibition rates were 8.40%,20.84%,34.90%,45.32%,55.38% after probucol with different concentrations acted at HRCEC for 24 hours,respectively.There was significant reduction in different concentrations of probucol treating groups at 24 hours in comparison with high glucose control group（P0.05）.There was no obvious statistical difference between the low glucose control group and high glucose control group（P=0.0670.05）.After acted at HRCEC for 24 hours of low glucose control group,high glucose control group and high glucose＋different concentrations（5 μmol·L-1,40 μmol·L-1,80 μmol·L-1） probucol groups,the differences between background gray values and positive gray values were 28.75±3.77,71.93±8.37,54.67±4.33,41.43±3.45,29.18±3.37,respectively.Compared with the high glucose control group,probucol decreased the intercellular expression of VEGF obviously,there was statistical difference（P0.05）,and which in dosage-dependant manner.However,in high glucose group,the expression of VEGF was significantly higher than that of low glucose control group（P0.05）.Conclusion Probucol can inhibit the proliferation and suppress the expression of VEGF of HRCEC in high glucose environment cultured in vitro.