糖尿病视网膜病变大鼠视网膜VEGF和PEDF的动态表达

Dynamic expressions of VEGF and PEDF in retina of rats with diabetic retinopathy

目的观察不同病程糖尿病(diabetic mellitus,DM)大鼠视网膜超微结构及视网膜血管内皮生长因子(vascular endothelial growth factor,VEGF)和色素上皮衍生因子(pigment epithelium-derived factor,PEDF)的表达情况,探讨VEGF、PEDF的表达在糖尿病视网膜病变中的变化及作用。方法取健康雄性SD大鼠76只,随机分为正常对照组(CON组)、DM 1个月组(DM1组)、DM 3个月组(DM2组)、DM 5个月组(DM3组),DM组大鼠实验前用链脲佐菌素制作DM模型。分别于造模后1个月、3个月、5个月用透射电镜观察大鼠视网膜超微结构,采用免疫组织化学染色、qRT-PCR和Western-blot观察视网膜VEGF和PEDF的表达。结果 DM初期大鼠视网膜细胞超微结构已出现改变,且随病程延长逐渐加重。免疫组织化学染色显示,CON组、DM1组、DM2组、DM3组视网膜VEGF的表达OD值分别为0.215±0.019、0.302±0.031、0.523±0.047、0.748±0.078,且其阳性染色逐渐加深;PEDF的表达OD值分别为0.908±0.073、0.735±0.087、0.607±0.055、0.187±0.039,阳性染色逐渐减弱。qRT-PCR显示,CON组、DM1组、DM2组、DM3组视网膜VEGF mRNA的相对表达量分别为0.105±0.014、0.372±0.037、1.031±0.095、2.364±0.198,PEDF mRNA的相对表达量分别为2.643±0.188、1.528±0.093、0.905±0.111、0.254±0.077。Western-blot显示,CON组、DM1组、DM2组、DM3组视网膜VEGF的蛋白表达量分别为0.328±0.028、0.436±0.034、0.651±0.028、0.793±0.026,PEDF的蛋白表达量分别为0.796±0.063、0.605±0.086、0.424±0.065、0.263±0.067。以上三项实验中组间差异均有统计学意义(均为P<0.05)。结论随DM病程延长,DM大鼠视网膜超微结构改变逐渐加重,VEGF表达不断降低。

Objective To observe the retinal ultrastructure of rats with diabetes mellitus（DM） and the expressions of vascular endothelial growth factor （VEGF） and pigment epithelium-derived factor（PEDF）. And to explore the expression changes and roles of VEGF and PEDF at different stages of diabetic retinopathy. Methods Seven- W-six male SD rats were randomly divided into CON group （the normal control group）, DM1 group（ rats with a DM history of 1 month） ,DM2 group（ rats with a DM history of 3 months） and DM3 group （ rats with a DM history of 5 months）. The DM model was es- tablished using streptozotocin before the experiment. At 1 month, 3 months and 5 months after modeling, respectively, the retinal ultrastructure of the rats was observed using transmission electron microscope; Retinal expressions of VEGF and PEDF were observed using immunohistochemical staining, qRT-PCR and Western-blot. Results The retinal ultrastructure changed at the preliminary stage of DM, and the condition worsened with the development of DM. According to immunohistochemical staining, OD values of the expression of VEGF in CON group, DM1 group, DM2 group and DM3 group were 0. 215 ± 0. 019,0. 302 ± 0. 031,0. 523 ± 0. 047 and 0. 748 ± 0. 078, and the positive staining gradually strengthened; OD values of the expression of PEDF were 0. 908 ± 0. 073,0.735 ±0.087,0.607 ±0.055 and 0.187 ±0.039 ,and the positive staining gradual- ly weakened. According to qRT-PCR, the respective relative expression level of retinal VEGF mRNA in the four groups were 0. 105 ±0. 014,0. 372 ±0. 037,1. 031 ±0. 095 and 2. 364 ± 0.198 ;The respective relative expression level of retinal PEDF mRNA in the four groups were 2.643 ± 0.188, 1. 528 ± 0.093,0. 905 ± 0.111 and 0.254 ± 0.077. According toWestern-blot, the respective protein expression level of VEGF in the four groups were 0.328 ± 0. 028 ,0. 435 ± 0. 034,0. 651 ± 0.028 and 0. 793 ± 0.025 ; The respective protein expression level of PEDF in the four groups were 0. 795 ± 0. 063,0. 505 ± 0. 086,0.424 ±0.055 and 0.253 ±0. 057. Differences among the three experimental groups present in all the three examina- tions were statistical significant（AU P 〈 0.05）. Conclusion As the course of DM developing,the retinal ultrastructure of the DM rats change for the worse gradually, the expression of VEGF gradually increase, while the expression of PEDF gradu- ally decrease.