CXCR4靶向shRNA质粒载体制备及体外鉴定.docVIP

CXCR4靶向shRNA质粒载体制备及体外鉴定[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Western blot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。 [关键词]黑色素瘤;趋化因子受体CXCR4;shRNA;质粒 [中图分类号]Q813.1[文献标识码]A [文章编号]1008-6455(2010)08-1167-04 Construction and in vitro identification of CXCR4 target plasmid vector of short hairpin RNA WU Bao-jin1,2,JIANG Hua2,LI Wen-peng3,WU Jian-ming2,ZHANG Ying-fan2,DING Wei1 (1.Department of Plastic Surgery,Huashan Hospital,Fudan University,Shanghai 200003,China; 2.Department of Plastic Surgery,Changzheng Hospital,the Second Military Medical University; 3. Department of Plastic Surgery,the Second Affiliated Hospital of Zhejiang University School of Medicine) Abstract:ObjectiveTo construct a CXCR4 specific shRNA recombinant plasmid vector and identify its inhibiting effect on the gene expression of CXCR4 in melanoma cell lines. MethodsA 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.ResultsCXCR4 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.ConclusionCXCR4-shRNA can be highly effectively transfected into melanoma cell, and induced post-transcriptional gene silenci