腺病毒介导的RNA干扰降低VEGF受体FLT-1,KDR转录抑制血管形成

Knockdown of FLT-1,KDR by Adenovirus-Delivered Small Interfering RNA Inhibits Angiogenesis

本研究用细菌内同源重组的方法构建了针对VEGF受体FLT-1,KDR的siRNA表达重组腺病毒Ad H1-siRNA/FLT-1和Ad H1-siRNA/KDR,感染HUVEC细胞,观察两种病毒对HUVEC细胞体外增殖和形成微血管的干扰作用.通过RT-PCR实验表明,Ad H1-siRNA/FLT-1和Ad H1-siRNA/KDR均可特异性地下调血管内皮细胞(HUVEC)中的FLT-1mRNA和KDR mRNA水平.与对照组相比,FLT-1的mRNA水平下降为40%,KDR的mRNA水平下降为52%.Ad H1-siRNA/FLT-1和Ad H1-siRNA/KDR对HUVEC细胞增殖均有干扰作用,加入病毒9 d后,对照组的细胞平均计数为31.5×104/μL,Ad H1-siRNA/FLT-1干扰病毒对照组为28×104/μL,Ad H1-siRNA/KDR干扰组为21.5×104/μL.Ad H1-siRNA/FLT-1和Ad H1-siRNA/KDR对HUVEC细胞在Matrigel上形成微血管均有干扰作用,对照组每HPF形成微血管数量为10.75条,Ad H1-siRNA/FLT-1干扰病毒对照组为10.25条,实验组Ad H1-siRNA/KDR每HPF形成微血管数量为7条.证明Ad H1-siRNA/FLT-1,Ad H1-siRNA/KDR均可干扰血管形成.

This study used the homologous recombination technique constructed a recombinant adenovirus AdH1-siRNA/FLT-1 and AdH1-siRNA/KDR targeting FLT-1 and KDR infects human endothelial cells （ HUVEC ） , observation the effect of the adenovirus interfere the proliferation of HUVEC in vitro and the tube formation. Reverse transcription-PCR showed that this adenovirus could specifically decrease level of the FLT-1 and KDR mRNA of HUVEC. Compared with control group,the FLT-1 and KDR mRNA expression of decreased to 40% and 52%. AdH1-siRNA/FLT-1 and KDR can efficiently interfere the proliferation of HUVEC,9 d after infection of adenovirus,the average cell number of control group and vector group are 31. 5×10^4/μL and 28×10^4/μL,that of groups respectively infected with AdH1-siRNA/VEGF are 21. 5×10^4/μL,and can efficiently interfere the tube formation of HUVEC on matrigel. Averagely,the tube number of every HPF is 10. 75 in the control group,and 10. 25 in the vector group. In the group treated with AdH1-siRNA/VEGF,there are 7 tubes in the HPE. It is sowed that AdH1-siRNA/FLT-1 and AdH1-siRNA/KDR can also interfere tube formation.