重组canstatin蛋白抑制体外视网膜微血管内皮细胞的迁移和增殖

Inhibition of recombinant canstatin protein on migration and proliferation of retinal microvascular endothelial cells in vitro

目的观察重组血管能抑素(canstatin)蛋白对体外培养的恒河猴视网膜微血管内皮细胞(RF/6A)迁移、增殖及磷酸化细胞外调节蛋白激酶(phospho-extracelluar regulated protein kinases,pERK)、磷酸化Akt表达的影响,初步探讨重组canstatin蛋白抑制视网膜新生血管的作用机制。方法体外培养RF/6A细胞系,在培养基中分别添加重组canstatin蛋白和等量的PBS,采用Transwell小室法检测各组发生迁移的内皮细胞并计数;铺Matri-gel胶,行体外成管实验,观察canstatin蛋白对内皮细胞成管的抑制作用;用MTT法检测重组canstatin蛋白对内皮细胞增殖的影响;Western blotting检测重组canstatin蛋白对血清刺激30min后RF/6A细胞pERK、磷酸化Akt蛋白表达的影响。结果加入重组canstatin蛋白后,显著抑制了细胞的迁移,重组canstatin蛋白组迁移的细胞数每视野(7.75±1.50)个显著低于PBS组(25.25±2.36)个(t=12.505,P=0.000);两组内皮细胞成管数分别为重组canstatin组每视野(18.67±7.02)个,也显著低于PBS组每视野(44.67±2.52)个(t=6.036,P=0.004)。与PBS组相比,重组canstatin蛋白组的内皮细胞的增殖也受到抑制,后者48h后平均吸光度A490为0.2869±0.0140,低于前者0.3349±0.0217(t=3.723,P=0.01)。同时,重组canstatin蛋白降低了RF/6A细胞中pERK蛋白的表达水平。结论重组canstatin蛋白能通过下调pERK蛋白的表达抑制RF/6A细胞的迁移和增殖,具有潜在抑制视网膜新生血管形成的作用。

Objective To explore the inhibiting mechanisms of recombinant canstatin protein on retinal neovascularization by observing the effects of recombinant canstatin protein on the migration and proliferation of retinal microvascular endothelial cells in vitro and the expression of phospho-extracelluar regulated protein kinases（pERK） and phosphorylation-Akt（pAkt）.Methods RF/6A cell lines were cultured in vitro.Recombinant canstatin protein and isochoric PBS were added to culture medium,respectively.Transwell chamber assay was used to examine and calculate the migratory endothelial cells.Matrigel was spread in canalization experiment in vtiro,which was taken to observe the inhibiting effects of canstatin protein on endothelial cell canalization,endothelial cell proliferation was assayed by MTT,and expression of pERK and pAkt in RF/6A cell lines were observed by Western blotting after stimulated with serum for 30 minutes.Results After added recombinant canstatin protein,cell migration was significantly inhibited.The number of migratory cells per visual field in compound canstatin group 7.75±1.50 was significantly lower than PBS group 25.25±2.36（t=12.505,P=0.000）,and also for number of cells with retinal canalization per visual field 18.67±7.02 compared with PBS group 44.67±2.52（t=6.036,P=0.004）.The proliferation of endothelial cells with adding canstain protein was inhibited compared with PBS group.After 48 hours,average absorbance A490 was 0.286 9±0.014 0 in compound canstatin group and 0.334 9±0.021 7 in PBS group（t=3.723,P=0.01）.Recombinant canstatin protein decreased the expression of pERK in RF/6A cell lines.Conclusions Recombinant canstatin protein can inhibit RF/6A cell migration and proliferation through decreasing the expression of pERK.And it has the potential to inhibiting retinal neovascularization.