CD147基因沉默对兔外周血单核巨噬细胞基质金属蛋白酶表达的影响

目的筛选出高效抑制兔外周血单核巨噬细胞中CD147表达的短链RNA(shRNA)慢病毒载体,观察CD147基因沉默后对基质金属蛋白酶(MMP)的影响。方法兔源CD147 mRNA序列,设计并构建shRNA慢病毒载体,实验分为空白组,阴性组,加CD147 shRNA慢病毒干扰依次为A组、B组、C组、D组,将其分别转染兔外周血单核巨噬细胞,72 h后观察转染效果,用RT-PCR测CD147 mRNA表达,ELISA法测CD147 MMP-2及MMP-9蛋白表达。结果 A、B、C、D组CD147 mRNA及CD147、MMP-2和MMP-9蛋白较空白组显著减少(P<0.01),A组CD147 mRNA和蛋白降低最显著,较空白组分别减少57.7%和50.9%(P<0.01),MMP-2、MMP-9蛋白表达分别减少95.9%和45.4%。结论成功构建并筛选出能高效、且特异阻断CD147基因表达的shRNA慢病毒载体。CD147基因沉默后,MMP-2和MMP-9表达明显减少,可能成为防治动脉粥样硬化新的治疗靶点。

Effect of CD147 gene silence on expression of matrix metalloproteinase in rabbit peripheral mononuclear-macrophages

Objective To study the effect of CD147 gene silence on expression of matrix metalloproteinase (MMP) in peripheral mononuclear-macrophages by screening the short chain RNA (shRNA)lentivirus vector that highly suppresses CD147 expression in rabbit peripheral mononuclear -macrophages.Methods A lentivirus vector was constructed according to the sequence of CD147 mRNA from rabbits.The animals were divided into blank control group,negative control group,and CD147 shRNA lentivirus intervention groups A-D.Transfection outcomes were observed by fluorescence microscopy 72 h after the rabbit peripheral mononuclear-macrophages were transfected into shRNA.Expressions of CD147 mRNA and protein,MMP-2 and MMP-9 were detected by fluorescence quantitative RT-PCR and ELISA,respectively.Results The expression levels of CD147 mRNA and protein,and CD147/MMP-2/MMP-9 were significantly lower in CD147 shRNA lentivirus intervention groups A-D than in blank control group(P<0.01).The expression levels of CD147 mRNA and protein,MMP-2 and MMP-9 protein were 57.7%and 50.9%,and 95.9%and 45.4%lower in CD147 shRNA lentivirus intervention group A than in blank control group(P<0.01).Conclusion A shRNA lentivirus vector is successfully constructed, which can effectively sreen and specifically block the CD147 gene expression.CD147 gene silence down-regulated expression of MMP-2 and MMP-9 may become a novel target for the prevention and treatment of atherosclerosis.