腺病毒载体介导的RNAi对胶质瘤U251细胞肝细胞生长因子受体表达的影响  

Effect of Adenovirus Vector-mediated RNA Interference on Expression of Hepatocyte Growth Factor Receptor c-Met in Glioma U251 Cells

作　　者：张红[1] 楚胜华[1] 冯东福[1] 马延斌[1] 邱建华[1] 朱志安[1] 

机构地区：[1]上海交通大学医学院附属第三人民医院神经外科,上海201900

出　　处：《中国临床神经外科杂志》2008年第7期412-415,共4页Chinese Journal of Clinical Neurosurgery

基　　金：上海高校选拔培养优秀青年教师科研专项基金项目(07JWYQ03);上海交通大学医学院优秀青年教师基金项目(07XYQ01)

摘　　要：目的探讨腺病毒载体介导的RNA干扰技术对胶质瘤细胞肝细胞生长因子(HGF)受体(c-Met)的表达和细胞凋亡的影响。方法用PCR法获得人U6启动子及带有c-Met反向互补靶序列的片段HU6shmet;利用腺病毒载体将其传递至U251细胞;分别采用RT-PCR和Westernblot方法检测U251细胞的c-MetmRNA和蛋白的表达,Annexin-V-PI双染法流式细胞术检测细胞的凋亡状况。结果获得了带有人U6启动子及c-Met反向互补靶序列的重组腺病毒载体rAdUshmet1和rAdUshmet2。转导了rAdUshmet1和rAdUshmet2的U251细胞的c-MetmRNA和蛋白相对表达量较未转导腺病毒及转导了rAdGFP和rAdU-sicon的U251细胞均有明显下降(P<0.01),rAdUshmet1和rAdUshmet2转导的U251细胞凋亡率分别为(13.5±4.2)%和(28.2±5.6)%,明显高于未转导腺病毒的及rAdGFP和rAdUsicon转导的U251细胞凋亡率(P<0.05)。结论腺病毒载体rAdUshmet通过抑制HGF受体c-Met表达,能在一定程度上阻断HGF-c-Met信号通路,有可能成为对胶质瘤进行基因治疗的有效载体。Objective To investigate the effects of Adenovurus vecror-mediated RNA intereference （RNAi） on the expression of hepatocyte growth factor （HGF） receptor, c-met in glioma U251 cells and apoptosis of glioma U251 cells. Methods Human U6 promotor and the HU6shmet, fragment with c-Met reverse complement target sequence were obtained by PCR. The Hu6shmet was packaged in adenovirus and transduced into U251 cells. The expressions of c-Met mRNA and-protein were determined respectively by RT-PCR and Western blot. The apoptosis of Glioma U251 cells was determined by flow cytometry. Results The Recombined adenovirus vector with human U6 promotor and c-Met reverse complement target sequence （named rAdUshmet 1 and rAdUshmet 2） were obtained. The c-Met mRNA and protein expression levels were significantly lower in the U251 cells transduced with the rAdUshmet 1 and rAdUshmet 2 than those in U251 cells without transduction with adenovirus, U251 cells transduced with recombined adenovirus with U6 small inteterence control（rAdUsicon） and U251 cells transduced with recombined adenovirus with green fluorescent protein（rAdGFP） （P〈0.01）. The apoptosis rates of the cells were （13.5±4.2）% and （28.2±5.6）% respectively in U251 cells transduced with rAdUshmet 1 and rAdUshmet 2, and were significantly higher than those in U251 cells without transduction with adenovirus, U251 cells transduced with rAdUsicon or with rAdGFP （P〈0.05）. Conclusions The rAdUshmet can inhibit c-Met expression and induce apoptosis of glioma cells, block the HGF-c-Met signal pathway partially and may act as an effective vector for gene therapy of glioma.

关 键 词：肝细胞生长因子受体 肝细胞生长因子 腺病毒 RNA干扰 

分 类 号：Q786[生物学—分子生物学] R739.41[医药卫生—肿瘤]