CXCR4靶向shRNA质粒载体的制备和体外鉴定  

Construction and in vitro identification of CXCR4 target plasmid vector of short hairpin RNA

作　　者：吴包金[1,2] 江华[2] 李文鹏[3] 吴建明[2] 张盈帆[2] 丁伟[1] 

机构地区：[1]复旦大学附属华山医院整形外科,上海200040 [2]第二军医大学附属长征医院整形外科,上海200003 [3]浙江大学医学院附属第二医院整形外科,浙江杭州31009

出　　处：《中国美容医学》2010年第8期1167-1170,共4页Chinese Journal of Aesthetic Medicine

基　　金：国家自然科学基金资助(30371472)

摘　　要：目的:设计并构建CXCR4靶向shRNA质粒载体,转染黑色素瘤细胞株,验证shRNA对黑色素瘤CXCR4基因的沉默效应。方法:设计合成CXCR4特异性shRNA,将其插入pSilencer质粒载体,并将重组后的pSilencer质粒载体经脂质体包裹转染黑色素瘤MV3细胞株,抗性筛选稳定抑制CXCR4表达的永久性克隆。应用RT-PCR技术检测shRNA对黑色素瘤细胞内CXCR4 mRNA表达,应用Westernblot法检测CXCR4蛋白变化。结果:成功构建和筛选出CXCR4特异性的shRNA质粒载体及稳定抑制CXCR4表达的永久性细胞克隆。阳性克隆测序结果表明合成的寡核苷酸链序列插入正确。稳定转染CXCR4-shRNA的高转移性黑色素瘤细胞MV3的CXCR4 mRNA和蛋白的表达较空白对照组明显下调。结论:脂质体介导shRNA体内表达法制备的CXCR4-shRNA在黑色素瘤细胞中可获得高效转染,并能产生特异性的基因沉默效应,以CXCR4为靶向的shRNA能够有效下调CXCR4基因的表达,有望为转移性黑色素瘤的靶向基因治疗提供一条新途径。Objective To construct a CXCR4 specific shRNA recombinant plasmid vector and identify its inhibiting effect on the gene expression of CXCR4 in melanoma cell lines.Methods A CXCR4 specific recombinant plasmid vector was prepared and transfected into the cultured MV3 cell line with lipofectamine 2000.RT-PCR and Western blot were used to detect the mRNA and protein expression of CXCR4,respectively.Results CXCR4 specific shRNA was constructed and transfected into the MV3 cell line.Agarose gel electrophoresis confirmed the vector had been inserted in the production of PCR,and sequencing result showed that composite CXCR4-shRNA inserting correctly in the positive clone.Both the CXCR4 protein and mRNA expression in the CXCR4-shRNA group were significantly lower than that of the control group.Conclusion CXCR4-shRNA can be highly effectively transfected into melanoma cell,and induced post-transcriptional gene silencing of CXCR4,which may become a potential target in the prevention and treatment of invasion and metastasis of melanoma.

关 键 词：黑色素瘤 趋化因子受体CXCR4 SHRNA 质粒 

分 类 号：Q813.1[生物学—生物工程]