RNA干扰技术对小鼠巨噬细胞基质金属蛋白酶9表达的抑制

Repressive effects of RNA interference technique on expression of matrix metalloproteinase-9 in macrophages of mice

目的:构建基质金属蛋白酶9靶向的RNA干扰质粒载体,观察其对小鼠巨噬细胞基质金属蛋白酶9基因表达的沉默作用。方法:实验于2004-11/2005-07在哈尔滨医科大学第一临床医学院中心实验室完成。设计能转录小发卡结构RNA的DNA序列,并与psiSTRIKETM质粒载体连接,构建受控于人RNA聚合酶Ⅲ启动子U6的真核表达载体,以脂质体法将重组质粒导入小鼠巨噬细胞内,采用Invitrogen公司LipofectamneTM2000转染试剂进行转染。实验分为实验组、阴性对照组和空白对照组。实验组每孔分别加入4μL脂质体和psiSTRIKETM/基质金属蛋白酶9重组质粒2μg;阴性对照组只加2μLLipofectamneTM2000转染试剂;空白对照组则不加任何干扰因素。分别于转染前、转染24,48,72h后用反转录-聚合酶链反应和免疫组织化学技术检测基质金属蛋白酶9mRNA及蛋白水平的表达情况。结果:①成功构建靶向基质金属蛋白酶9基因发夹状RNA干扰质粒载体。②实验组转染重组质粒24,48,72h后阳性细胞率与转染前相比逐渐减少(分别为70%,42%,32%,99%,P<0.01),而阴性对照组和空白对照组则差异无显著性意义。③转染小鼠巨噬细胞后,基质金属蛋白酶9mRNA蛋白表达下调。结论:构建的RNA干扰真核表达载体能明显抑制基质金属蛋白酶9mRNA及蛋白的表达,提示通过减少动脉粥样硬化斑块细胞外基质的降解,来稳定动脉粥样硬化斑块,在基因治疗方向为斑块稳定进行了新的尝试。

AIM： To construct plasmid vector of RNA interference specific for matrix metalloproteinases （MMP）-9 and observe the silencing effect on gene expression of MMP-9 in macrophage of mice. METHODS： The experiment was conducted in the Central Laboratory of First Clinical Medical College of Harbin Medical University from November 2004 to July 2005. The sequences of transcribed small hairpin RNAs （shRNA） RNA and DNA were designed, and connected with plasmid psiSTRIKE^TM, which is the eukaryotic expression vector controlled by the U6 promoter of RNA polymerase Ⅲ, and cell line were transfected with recombinant plasmid by Lipofectamne^TM 2000 manufactured by Invitrogen Corporation. The experiment was divided into experiment group, negative control group and blank control group. 4 μL of lipoplast and psiSTRIKE^TM /2 μg of MMT-9 recombinant plasmid were added into each pore of experiment group. 2 μL of Lipofectamne^TM 2000 transfection was added into negative control group, while the blank control group was interfered by no factors. RT-PCR and immunohistochemical method were used to detect the expression of mRNA in MMP-9 as well as the protein respectively before transfection and 24, 48, 72 hours after transfection. RESULTS： （1）The recombinant plasmid of RNA interference specific for MMP-9 Was successfully constructed. （2） The positive cell rate in recombinant plasmid of experiment group at 24, 48 and 72 hours after transfection was gradually decreased than those before transfection （respectively were 70%, 42%, 32% and 99%, P 〈 0.01）, while there were no significant differences between negative control group and blank control group. （3）The expression of MMP-9 mRNA protein was down-regulated in macrophage of rats after transfection. CONCLUSION： Reconstructed RNA eukaryotic expression vector can significantly inhibit the expressions of MMP-9 mRNA and protein, which indicate that to stabilize the atherosclerotic plaque by decrease the degradation of extracellular matrix of atherosclerotic plaque lays the basis for its application in the treatment of unstable plaque.