pcDNA3.1/hTIMP-1-EGFP真核表达载体的构建及其在血管平滑肌细胞中的表达

Construction of Eukaryotic Expression Plasmid of pcDNA3.1/Human Tissue Inhibitor of Metalloproteinase-1-Enhanced Green Fluorescent Protein and Its Expression in Vascular Smooth Muscle Cells

目的构建标记有增强型绿色荧光蛋白(EGFP)的人基质金属蛋白酶组织抑制因子-1(hTIMP-1)真核表达质粒pcDNA3.1/hTIMP-1-EGFP,并通过转染大鼠血管平滑肌细胞(SMCs)验证hTIMP-1的表达。方法将hTIMP-1及编码EGFP的互补脱氧核糖核酸(cDNA)序列通过扩增、酶切、插入pcDNA3.1质粒的多克隆位点,构建pcDNA3.1/hTIMP-1-EGFP真核表达质粒;应用脂质体介导的基因转染技术,将基因导入SMCs(pcDNA3.1/hTIMP-1-EGFP转染组)。应用荧光显微镜观察EGFP的表达情况;流式细胞仪检测转染效率;逆转录-聚合酶链反应(RT-PCR)、蛋白免疫印迹法(Western blotting)等方法检测hTIMP-1的表达情况;明胶酶谱法检测基质金属蛋白酶MMP-2、MMP-9活性。用空质粒pcDNA3.1转染SMCs(空质粒pcDNA3.1转染组)和未转染质粒的SMCs(未转染组)作为对照。结果pcDNA3.1/hTIMP-1-EGFP转染组重组质粒转染SMCs后,SMCs生长受到抑制;转染24h后在荧光显微镜下可见强绿色荧光,流式细胞仪检测转染效率约为15%;RT-PCR检测结果显示,SMCs出现646 bp目的基因;Western blotting检测显示,在转染后的血管SMCs中有hTIMP-1表达;明胶酶谱法检测结果显示,MMP-2、MMP-9活性降低;与空质粒pcDNA3.1转染组和未转染组比较差异有统计学意义(P<0.05)。结论TIMP-1真核表达质粒的构建和在血管SMCs中的表达,为TIMP-1基因治疗的研究奠定了基础。

Objective To generate a eukaryotic expression plasmid-pcDNA3. 1/human tissue inhibitor of metalloproteinase-1 （hTIMP-1）-enhanced green fluorescent protein （EGFP）, carrying hTIMP-1 and labeled with EGFP, and to examine the expression of hTIMP-1 in vascular smooth muscle cells （SMCs） transferred with hTIMP. Methods The recombinant plasmids of pcDNA3. 1/hTIMP-1-EGFP were obtained by-polymerase chain reaction （PCR） amplification, splicing, and insertion of complementary deoxyribonucleic acid （cDNA） fragments of hTIMP-1 and EGFP. The target gene was transferred to the primarily cultured SMCs （pcDNA3. 1/hTIMP-1-EGFP transferred group ） by using cationic liposome mediated gene transfection technique. EGFP expression was detected by fluorescence microscopy, and the transfection rate was determined by flow cytometry. Reverse transcriptase po｜ymerase chain reaction （RT-PCR）, Western blotting, and other techniques were used to detect the expression of hTIMP-1 gene. The biological activity of matrix metalloproteinase-2 （MMP-2） and matrix metalloproteinase-9 （MMP-9） were studied by zymographic analysis of gelatinases. Blank plasmid-pcDNA3.1 transferred SMCs （blank plasmid pcDNA3. 1 transferred group） and untransferred SMCs （untransferred group） were used as control. Results In pcDNA3. 1/hTIMP-1-EGFP transferred group, the growth ability of SMCs was profoundly inhibited, bright green fluorescence was observed by fluorescence microscopy 24 hours after transfection in SMCs,the rate of transfection analyzed with flow cytometry was 15%, RT-PCR results showed that the genome of hTIMP-1 transferred SMCs contained a 646 bp specific fragment of hTIMP-1 gene, Western blotting results proved hTIMP 1 protein expression in SMCs transferred by hTIMP-1, zymographic analysis of gelatinases showed decreased activity of MMP-2 and MMP-9, compared to those in blank plasmid-pcDNA3. 1 transferred group and untransferred group, significant differences were observed （P〈0. 05）. Conclusion The generation of a eukaryotie expression plasmid carrying TIMP-1 gene and its expression in SMCs provide a sound basis for hTIMP-1 gene therapy.