基质金属蛋白酶-1shRNA真核表达载体的构建

目的 构建基质金属蛋白酶-1(MMP-1)特异的RNA干扰质粒载体.方法 根据GenBank 数据库提供的MMP-1基因核苷酸序列,选择设计能转录短发夹状RNA (Short hairpin RNAs,shRNA) 的DNA 序列,并与pWH1质粒载体连接,用酶切的方法 进行鉴定;将构建成功的特异性表达载体(pWH1-MMP1)稳定转染至人腺样囊性癌ACC-M细胞系后,应用RT-PCR、免疫组织化学及Western Blot对细胞MMP1的表达进行检测.结果 在成功转染pWH1-ACC-M表达载体的ACC-M细胞中,MMP1 mRNA和蛋白的表达明显降低.结论 成功构建了MMP1特异性shRNA表达载体,可有效地沉默MMP1基因,为进一步研究MMP1表达与腺样囊性癌增殖和转移的相关性奠定了一定的实验基础.

Construction of eukaryotic expression vectors of short hairpin RNA for MMP1

Objective To construct eukaryotic expression vectors of RNA interference specific to matrix metalloproteinase 1（MMP1）.Methods Genome sequences of MMP1 gene were retrieved from Genbank and cDNA that was capable of coding expression of shRNA（small hairpin RNAs） was designed for MMP1 gene.The cDNA was synthesized and inserted into plasmid pWH1,and the recombinant pWH1-MMP1 expression vector was identified using enzyme cutting.Then,pWH1-MMP1 expression vector was transfected into salivary adenoid cystic carcinoma ACC-M cells.Finally,the expression of MMP1 in ACC-M cells transfected with pWH1-MMP1 expression vector was evaluated.Results Compared with ACC-M cells and ACC-M cells transfected with plasmid pWH1,the ACC-M cells transfected with pWH1-MMP1 expression vector showed a low mRNA and protein expression of MMP1.Conclusions The constructed MMP1 shRNA expression vector can block the expression of MMP1 in salivary adenoid cystic carcinoma cells,potentially facilitating further studies on the significance of MMP1 in the proliferation,metastasis and experimental treatment of salivary adenoid cystic carcinoma.