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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 investigate the effect of recombinant canstatin on the migration and proliferation of rhesus monkey retinal capillary endothelial cells (RF / 6A) cultured in vitro and the phosphorylation of phospho-extracellular regulated protein kinase (pERK) Akt expression, and to explore the mechanism of action of recombinant canstatin protein on retinal neovascularization. Methods The RF / 6A cell line was cultured in vitro. Recombinant canstatin protein and PBS were added into culture medium. Transwell chamber assay was used to detect the number of endothelial cells that migrated in each group. Matri-gel gel was used to conduct the in vitro tube formation test The inhibitory effect of canstatin on endothelial cells was observed. The effect of recombinant canstatin protein on the proliferation of endothelial cells was detected by MTT assay. The expression of pERK and phospho-Akt protein in RF / 6A cells was detected by Western blotting influences. Results The migration of canstatin protein was significantly inhibited by the addition of recombinant canstatin protein (7.75 ± 1.50 per field of field), which was significantly lower than that of PBS group (25.25 ± 2.36) (t = 12.505, P = 0.000) (18.67 ± 7.02) per field of view in the canstatin group and 44.67 ± 2.52 per field of view in the PBS group (t = 6.036, P = 0.004), respectively. Compared with the PBS group, the proliferation of endothelial cells in the recombinant canstatin protein group was also inhibited. The average absorbance A490 after 48h was 0.2869 ± 0.0140, lower than the 0.3349 ± 0.0217 (t = 3.723, P = 0.01). Concurrently, recombinant canstatin reduced the expression of pERK protein in RF / 6A cells. Conclusion Recombinant canstatin protein can inhibit the migration and proliferation of RF / 6A cells by down-regulating the expression of pERK protein and potentially inhibit retinal neovascularization.