变性Ⅰ型胶原对内皮细胞增殖、迁移及血管形成相关蛋白合成的影响

Effect of denatured collagen type Ⅰ on endothelial cell proliferation, migration and angiogenesis-related proteins expression

目的·探讨变性Ⅰ型胶原对内皮细胞增殖、迁移及血管生成相关蛋白合成的影响。方法·人脐静脉内皮细胞传代培养后分别接种于正常胶原(正常胶原组)、半量正常胶原(半量胶原组)和变性胶原(变性胶原组)包被的培养板上,2 d后用CCK-8法检测细胞增殖活性,划痕实验观察细胞迁移能力,Western blotting检测膜型基质金属蛋白酶-1(membrane-type 1 matrix metalloproteinase,MT1-MMP)和血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)蛋白表达水平,ELISA法检测血管生成素1(angiopoietin 1,Ang-1)和Ang-2的蛋白分泌情况。结果·CCK-8法检测结果显示,变性胶原组细胞的增殖活性显著高于正常胶原组和半量胶原组(均P<0.05);划痕实验结果显示,变性胶原组细胞的迁移能力最强,16 h即可将划痕基本完全覆盖。变性胶原组MT1-MMP和VEGF表达水平均明显高于其他2组(均P<0.05),Ang-2蛋白分泌量显著高于正常胶原组(P<0.05);但Ang-1分泌与其他2组差异无统计学意义(P>0.05)。结论·变性Ⅰ型胶原能促进内皮细胞增殖、迁移,增加MT1-MMP、VEGF、Ang-2的表达,提示变性胶原在创面修复的血管化过程中可能有积极作用。

Objective·To reveal the effect of denatured collagen typeⅠon the endothelial cell proliferation,migration,and angiogenesis-related proteins expression.Methods·Human umbilical vein endothelial cells were cultured on the plates coated with normal collagen(normal collagen group),half concentration normal collagen(half collagen group)or denatured collagen(denatured collagen group).CCK-8 assay was performed to test cell proliferation ability two days later.The effect of collagen on cell migration was measured by scratch test.The expressions of membrane-type 1 matrix metalloproteinase(MT1-MMP)and vascular endothelial growth factor(VEGF)were detected by Western blotting.The expressions of angiopoietin 1(Ang-1)and Ang-2 were measured by ELISA.Results·CCK-8 results showed that the proliferation activity of denatured collagen group was significantly higher than that of normal collagen group and half collagen group(P<0.05).Cell migratory capacity was increased in denatured collagen group,of which the scratch was almost covered after 16 h.The protein expressions of MT1-MMP and VEGF were significantly higher in denatured collagen group than those in the other two groups(P<0.05),in which the concentration of Ang-2 was also higher than that in normal collagen group(P<0.05).However,no significant difference was found in the concentration of Ang-1 among the three groups.Conclusion·Denatured collagen typeⅠcan promote proliferation,migration and MT1-MMP,VEGF,and Ang-2 expressions in endothelial cells,suggesting that collagen denaturation may play an active role in the process of angiogenesis during wound healing.