LOX-1对ox-LDL诱导的小鼠巨噬细胞MMP-9表达的影响

Effect of LOX-1 on expression of MMP-9 in macrophages induced by ox-LDL

目的观察LOX-1对ox-LDL诱导的小鼠巨噬细胞MMP-9表达的影响。方法体外培养小鼠单核巨噬细胞RAW264. 7,构建LOX-1小干扰RNA(LOX-1-siRNA),并用LOX-1-siRNA 0. 5μg、1μg、1. 5μg与脂质体转染剂(μl)比例为1∶1,1∶2,1∶3转染RAW264. 7细胞48 h,经荧光显微镜观察不同转染条件下的转染效率,并分别使用反转录-多聚酶反应(RTPCR)和Western blot的方法检测各组LOX-1的mRNA和蛋白表达水平并选取最佳转染比例。随机将RAW264. 7细胞分成4组:对照组、ox-LDL组、ox-LDL+LOX-1-siRNA组和p Genesil组;分别使用反转录-多聚酶反应(RT-PCR)和Western blot的方法检测各组MMP-9的mRNA和蛋白表达水平。结果 (1)LOX-1-siRNA1及LOX-1-siRNA2分别转染RAW264. 7细胞48 h,LOX-1-siRNA2抑制作用强于LOX-1-siRNA1。(2)用LOX-1-siRNA与脂质体转染剂不同比例转染RAW264. 7细胞48 h后,当LOX-1-siRNA量为1. 0μg、Lipofectamine2000的量为2μl时转染效率最高,与其余任一组相比差异有统计学意义(P <0. 05)。(3)与对照组比较,ox-LDL组MMP9 mRNA表达水平明显升高; ox-LDL+LOX-1-siRNA组与ox-LDL组比较MMP-9 mRNA表达明显减弱; p Genesil组MMP-9 mRNA表达与对照组比较差异无统计学意义。(4)与对照组相比,ox-LDL组MMP9蛋白表达明显增加; ox-LDL+LOX-1-siRNA组与ox-LDL组比较MMP-9蛋白表达明显减弱; p Genesil组MMP-9蛋白表达与对照组比较差异无统计学意义。结论 ox-LDL可促进小鼠巨噬细胞MMP9的表达;抑制小鼠巨噬细胞LOX-1的表达后,ox-LDL促进MMP9的表达相应减弱。由此可见ox-LDL可通过LOX-1受体促进MMP-9表达。

Objective To investigate the effect of LOX-1on the expression of MMP-9in macrophages induced by ox-LDL.Methods Mouse mononuclear macrophage RAW264.7was cultured in vitro,and LOX-1small interfering RNA(LOX-1-siRNA)was constructed.RAW264.7cells were transfected with LOX-1-siRNA(μg)/Lipofectamine2000(μl)at the ratios of1∶1,1∶2,1∶3 for 48h.The transfection efficiency was observed by fluorescence microscope.The expression of LOX-1mRNA and protein was respectively detected by RT-PCR and Western blot to select the best transfection ratio.RAW264.7cells were randomly divided into four groups:control group,ox-LDL group,ox-LDL+LOX-1group,and pGenesil group.The mRNA and protein expression levels of MMP-9were detected using RT-PCR and Western blot.Results①After transfected into RAW264.7 cells for48h,the inhibition efficacy of LOX-1-siRNA2 was stronger than that of LOX-1-siRNA1.②The transfection efficiency of RAW264.7 cells was the highest after transfection for48h with LOX-1-siRNA of 1.0 μg and Lipofectamine 2000 of 2 μl,and higher than that of the other ratios(P<0.05).③The expression of MMP-9mRNA in ox-LDL group was significantly higher than that in control group,and the expression of MMP-9mRNA in ox-LDL+LOX-1-siRNA group was significantly lower than that in ox-LDL group.There was no significant difference in MMP-9mRNA expression between pGenesil group and control group.④The expression of MMP-9protein in ox-LDL group was significantly higher than that in control group,and the expression of MMP-9protein in ox-LDL+LOX-1-siRNA group was significantly lower than that in ox-LDL group.There was no significant difference in MMP-9mRNA expression between pGenesil group and control group.Conclusion The ox-LDL can promote the expression of MMP-9 in mouse macrophages,and the expression of MMP-9 in mouse macrophages can be decreased by ox-LDL after LOX-1is inhibited,which suggests that ox-LDL can promote the expression of MMP-9through LOX-1receptor.