VEGFR_2siRNA腺病毒载体的构建及其对人结肠癌细胞系的影响

Construction of adenoviral vector encoding VEGFR_2siRNA and its effect on human colorectal cancer LOVO cell line

目的:构建血管内皮细胞生长因子受体2(VEGFR2)siRNA腺病毒载体,并研究其对结肠癌细胞系LOVOVEGFR2表达的影响.方法:首先构建含有VEGFR2siRNA的鼠U6载体(mU6-VEGFR2siRNA);利用HindⅢ与XbaⅠ双酶切将鼠U6载体启动子和VEGFR2siRNA克隆入无启动子穿梭载体promoterlesspShuttlevector,得到pShuttle-mU6pro-VEGFR2siRNA;将pShuttle-mU6pro-VEGFR2siRNA与腺病毒骨架质粒(pAdeasy-1)在BJ5183细菌中进行同源重组,得到PAdeasy-mU6Pro-VEGFR2siRNA;利用得到的重组腺病毒感染人结肠癌LOVO细胞系,观察VEGFR2siRNA对结肠癌细胞VEGFR2表达的影响.采用West-blot检测感染前后VEGFR2蛋白表达水平的变化.结果:成功构建血管内皮细胞生长因子受体2(VEGFR2)siRNA腺病毒载体,该载体显著抑制LOVO细胞中VEGFR2的蛋白表达水平(P<0.01).结论:构建的Adeasy-mU6pro-VEGFR2siRNA腺病毒能有效地抑制LOVO细胞中VEGFR2表达,从而为肿瘤的基因治疗打下基础,也为肿瘤血管抑制治疗提供了新思路.

AIM: To construct an adenovirus vector for efficient delivery of vascular endothelial growth factor receptor 2 (VEGFR2) siRNA, and to investigate its effect on human colorectal cancer LOVO cells. METHODS: After digestion with Hind Ⅱ and Xba I, the U6 promoter and VEGFR2siRNA were cloned into the promoterless shuttle vector to construct pShuttle-mU6pro-VEGFR2SiRNA. Then the product vector was cotransformed with adenovirus backbone containing plasmid pAdeasy-1to produce pAdeasy-mU6pro-VEGFR2siRNA by homologous recombinantion. Finally the human colorectal cancer LOVO cells were infected with the recombinant adenovirus and the VEGFR2expression was examined using Western-blot. RESULTS: The pAdeasy-mU6pro-VEGFR2siRN A was successfully constructed. VEGFR2siRNA mediated by the adenovirus significantly inhibited the expression of VEGFR2 in LOVO cell lines(P<0.01). CONCLUSION: The pAdeasy-mU6pro-VEGFR2siRNA can effectively down-regulate VEGFR2 expression in LOVO cell line, which lays the foundation for gene therapy of colorectal cancer.