RGD-TAT-KDRsiRNA慢病毒载体构建及抗肿瘤活性  

Lentiviral vector construction of RGD-TAT-KDR siRNA fusion gene and assessment of its antitumor activity

作　　者：张俭丽[1] 郑旭[2] 王杰[3] 

机构地区：[1]沈阳医学院附属卫生学校基础护理教研室,辽宁沈阳110034 [2]沈阳市和平区疾病预防控制中心 [3]沈阳医学院继续教育学院

出　　处：《中国公共卫生》2015年第5期594-597,共4页Chinese Journal of Public Health

基　　金：辽宁省自然科学基金(201102214);沈阳市科技局科技计划项目(F11-264-1-29)

摘　　要：目的构建精氨酰-甘氨酰-天冬氨酸(RGD)介导的穿膜肽(TAT)-血管内皮细胞生长因子受体(KDR)siRNA融合基因慢病毒载体,探讨其对人肺癌A549细胞的抑制效果。方法设计合成编码RGD的2条寡核苷酸链,克隆到p GC/TAT-KDR siRNA载体的TAT下游,构建TAT-RGD-KDR siRNA融合基因载体;慢病毒包装并感染A549细胞;通过Western blot和real-time PCR检测A549细胞KDR基因表达水平;通过噻唑蓝法、双色法流式细胞仪和Transwell侵袭实验检测KDR基因沉默对A549细胞凋亡和侵袭力的影响。结果测序和Blast比对证实重组载体中的RGD序列与设计一致;TAT-RGD-KDR siRNA融合基因慢病毒载体转染A540细胞KDR mRNA和蛋白表达水平分别为(22.7±3.9)%和(19.3±2.7)%,明显低于TAT-KDR siRNA融合基因慢病毒载体转染组,差异有统计学意义(P<0.01);该载体具有较强的抑制A549细胞增殖、促进细胞凋亡和抑制细胞体外侵袭力的作用。结论 TAT-RGD-KDR siRNA融合基因慢病毒载体通过抑制细胞KDR mRNA和蛋白表达,抑制A549细胞增殖和侵袭、促进细胞凋亡。Objective To construct a lentiviral vector of transcriptional activator protein-kinase insert domain receptor（ TAT-KDR） siRNA fusion gene mediated by arginyle-glycyl-d-aspartic acid（ RGD） and to assess its effects on human lung cancer A549 cells. Methods Tw o oligonucleotide chains of coding RGD were synthetized and then cloned into dow nstream of thrombin-antithrombin III complex（ TAT） in p GC / TAT-KDR siRNA vector to construct TAT-RGD-KDR siRNA fusion gene vector. Lentivirus was packaged and used to infect A549 cells. The expression level of KDR gene in A549 cells was detected with Western blot and real-time PCR. The proliferation,apoptosis and invasion ability of A549 cells were measured with 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide（ M TT） method,dual color flow cytometry,and transw ell invasion assay. Results Sequencing and Basic Local Alignment Search Tool（ BLAST） alignment confirmed that the RGD sequence was consistent with the design of the recombinant vector and the vector could reduce expression level of KDR mRNA and protein in A549 cells and inhibit the proliferation and invasiveness and promote apoptosis of A549 cells in vitro. Conclusion The lentiviral vector of TAT-RGD-KDR siRNA fusion gene can inhibit the proliferation and invasion and promote the apoptosis of A549 cell line by inhibiting the expression level of KDR mRNA and protein.

关 键 词：精氨酰-甘氨酰-天冬氨酸(RGD) 穿膜肽(TAT) 血管内皮细胞生长因子受体(KDR) TAT-RGD-KDR siRNA融合基因 慢病毒载体 

分 类 号：R373.9[医药卫生—病原生物学]