MMPs激动剂和抑制剂对人巩膜成纤维细胞MMP-1/2以及Ⅰ型胶原表达的作用研究

Effect of agonists and inhibitors of matrix metalloproteinases on expression of matrix metalloproteinase 1/2 and collagen Ⅰ in human scleral fibroblast

目的研究基质金属蛋白酶(MMPs)家族MMP-1、MMP-2激动剂和抑制剂对人巩膜Ⅰ型胶原表达的作用,探讨MMP-1和MMP-2在人巩膜胶原代谢中的作用。方法应用人巩膜成纤维细胞系作为研究模型,用荧光定量检测MMPs激动剂醋酸氨基苯汞(APMA)及抑制剂对人巩膜成纤维细胞MMP-1和MMP-2活性的影响;实时荧光定量反转录聚合酶链反应以及免疫印迹蛋白定量法检测比较激动剂组与抑制剂组对MMP-1/2以及Ⅰ型胶原mRNA和蛋白表达的变化;最后用siRNA干扰敲除MMP-1、MMP-2或MMP-1/2后,检测巩膜成纤维细胞Ⅰ型胶原蛋白水平的变化。结果 MMPs激动剂引起MMP-1/2活性显著增加,Ⅰ型胶原mRNA和蛋白表达下降,MMPs抑制剂引起MMP-1/2活性显著下降,相应的Ⅰ型胶原mRNA和蛋白表达增加(均P<0.01)。激动剂可引起MMP-1mRNA表达增加(P<0.01),对MMP-2mRNA表达无明显影响(P>0.05),而抑制剂能引起MMP-2mRNA表达下降(P<0.01),但对MMP-1mRNA表达无显著影响(P>0.05)。应用siRNA干扰方法敲除MMP-1、MMP-2或两者同时被敲除后,MMP-1蛋白表达均显著降低(均P<0.01),MMP-2除在MMP-1siRNA干扰敲除组无明显变化外(P>0.05),其余各组均显著下降(均P<0.01);Ⅰ型胶原的表达在各敲除组中显著增加,当MMP-1和MMP-2均干扰敲除时,Ⅰ型胶原的表达增加最为显著(均P<0.01)。结论在人巩膜成纤维细胞系中,MMP-1/2激动剂和抑制剂均能引起MMP-1/2活性改变及相应的MMP-1/2蛋白表达变化,并最终导致Ⅰ型胶原表达的变化;MMP-1和MMP-2是调控人巩膜Ⅰ型胶原重塑的关键因子。

Objective To investigate the effect of agonist and inhibitor of matrix metalloproteinase-1 （ MMP-1 ） and MMP-2 in matrix metalloproteinases （MMPs） on the expression of type I collagen and the effect of MMP-1 and MMP-2 in the development of human scleral collagen. Methods Human scleral fibroblast line was established as a model. Fluorescent quantitative assay was used to investigate the effect of P-aminophenylmercuric acetate （APMA） and inhibitors of MMP-1 and MMP-2 on MMP-1 and MMP-2 in human scleral fibroblast. Real-time PCR （RT-PCR） and Western-blot were used to compare the expression of mRNA and protein levels of MMP-1 ,MMP-2 and type I collagen after exposure to APMA and inhibitors of MMP-1/2. Finally, Gene of MMP-1 or MMP-2 or both were knocked out with small interfering RNA （siRNA） and the expression of type I collagen was detected. Results APMA significantly improved the activity of MMP-1/2 and decreased the expression of type I collagen on both mRNA and protein levels （ P 〈 0.01 ） ; inhibitors of MMPs decreased the activity of MMP-1/2 and increased the expression type I collagen on both mRNA and protein levels（ P 〈 0. O1 ）. APMA increased the expression of mRNA in MMP-1 （P 〈 0.01 ） but not MMP-2 （P 〉 0.05 ）. When gene of MMP-1 or MMP-2 or both were knocked out with siRNA, the protein levels of MMP-1 were significantly decreased （P 〈 0.01 ） ; MMP-2 were also significantly decreased （ P 〈 0.01 ） except in the MMP-1 knockdown group （P 〉0.05）; Type I collagen were significantly increased（ P 〈 0.01 ） especially when the MMP-1 and MMP-2 were both knocked out. Conclusions In human scleral fibroblast line, both agonist and inhibitor could change the activity and the protein level of MMP-1 and MMP-2 and finally change the expression of type I collagen. MMP-1 and MMP-2 were key factors in rebuilding of type I collagen in human sclera. （Chin J Ophthalmol and Otorhinolaryngo1,2013,13：91-95 ）