TNF-α和机械牵拉对圆锥角膜成纤维细胞MMP/TIMP及IL-6表达的影响

Effects of TNF-αand Mechanical Stretch on MMP/TIMP Ratios and the Expression of IL-6 in Keratoconus Corneal Fibroblasts

研究肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)与机械牵拉对圆锥角膜成纤维细胞白细胞介素-6(Interleukin-6,IL-6)、基质金属蛋白酶-1,-3(matrix metalloproteinase-1,-3,MMP-1,-3)、组织基质金属蛋白酶抑制剂-1,-2(tissue inhibitors of metalloproteinase-1,-2,TIMP-1,-2)基因表达与蛋白合成的影响。对体外培养的圆锥角膜成纤维细胞进行牵拉幅度12%、频率0.1Hz的周期性牵拉6h,并给予1ng/mL TNF-α处理,采用Real-Time PCR技术检测细胞中IL-6,MMP-1,MMP-3,TIMP-1,TIMP-2mRNA的表达,ELISA方法检测蛋白的含量。TNF-α单独作用可以诱导圆锥角膜成纤维细胞IL-6、MMP-1以及MMP-3的基因表达与蛋白合成(p<0.05),对TIMP-1、TIMP-2的基因表达与蛋白合成无明显影响,提高MMP-1/TIMP-2以及MMP-3/TIMP-2蛋白浓度的比值(p<0.05);机械牵拉单独作用促进IL-6、MMP-1以及MMP-3的基因表达与蛋白合成(p<0.05),下调TIMP-1、TIMP-2的基因表达与蛋白合成(p<0.05),上调MMP/TIMP蛋白浓度的比值(p<0.05);两者联合作用则表现出协同效应(p<0.05).提示TNF-α和机械牵拉可能通过上调IL-6及MMP/TIMP的比例促进圆锥角膜基质降解,加剧角膜组织破坏,导致角膜膨隆的发生发展。

Explored were the effects of tumor necrosis factor-α(TNF-α)and mechanical stretch on the expression of interleukin-6(IL-6),matrix metalloproteinase-1,-3(MMP-1,MMP-3),and tissue inhibitors of metalloproteinase-1,-2(TIMP-1,TIMP-2)in keratoconus corneal fibroblasts.Isolated keratoconus corneal fibroblasts were subjected to 12% equibiaxial stretch at frequency of 0.1 Hz for 6 hin the presence of 1 ng/ml TNF-α.The gene expressions of IL-6,MMP-1,-3,and TIMP-1,-2 were detected by real-time polymerase chain reaction(RealTime PCR).Culture supernatants were assayed by enzyme-linked immunosorbent assay(ELISA)for IL-6,MMP-1,-3,and TIMP-1,-2.TNF-αinduced IL-6,MMP-1,MMP-3 mRNA expression and protein synthesis(p<0.05),had no significant influence on the expression of TIMP-1,-2,and increased concentration ratios of MMP-1/TIMP-2 and MMP-3/TIMP-2 in keratoconus corneal fibroblasts(p<0.05);Cyclic stretch induced IL-6,MMP-1,MMP-3 mRNA expression and protein synthesis(p<0.05),inhibited expression of TIMP-1,-2(p<0.05),and increased concentration ratios of MMP/TIMP in keratoconus corneal fibroblasts(p<0.05);TNF-αand stretch had a synergistic effect.Mechanical stretch combined with TNF-αmay facilitate the corneal tissue destruction by upregulation of IL-6 and MMP/TIMP ratios,thereby contribute to the development of keratectasia.