腺病毒介导的RNA干扰降低VEGF转录抑制血管形成的研究

Study on Inhibitation of the Tube Formation by RNA Introduced by Adenovirus by its Interference With and Decrease of VEGF Transcription

用细菌内同源重组的方法构建了针对VEGF的siRNA表达重组腺病毒AdH1-siRNA/VEGF,感染HUVEC细胞,观察该病毒对HUVEC细胞体外增殖和形成微血管的干扰作用.通过Real-timeRT-PCR实验表明,AdH1-siRNA/VEGF可特异性地下调血管内皮细胞(HUVEC)中的VEGFmRNA水平.与对照组相比,VEGF的mRNA水平下降为50%,AdH1-siRNA/VEGF对HUVEC细胞增殖有干扰作用.加入病毒9d后,对照组的细胞平均计数为31.5×104/μL,干扰病毒对照组为28×104/μL,AdH1-siRNA/VEGF干扰组为21.5×104/μL.AdH1-siRNA/VEGF对HUVEC细胞在Matrigel上形成微血管有干扰作用,对照组每HPF形成微血管数量为10.75条,干扰病毒对照组为10.25条,实验组AdH1-siRNA/VEGF每HPF形成微血管数量为7条.证明AdH1-siRNA/VEGF可以干扰血管形成.

This study uses the homologous recombination technique to construct a recombinant adenovirus AdH1-siRNA/VEGF targeting VEGF infects human endothelial cells （ HUVEC）, observes the interference of the adenovirus with the proliferation of HUVEC in vitro and the tube formation. The experiement of real-time reverse transcription- PCR shows that this adenovirus can especially decrease level of the VEGF mRNA of HUVEC. Compared with control group,the mRNA expression of VEGF decreased to 50%. AdH1-siRNA/VEGF can efficiently interfere the prolifeyation of HUVEC. Nine days 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 respectively, 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 Matyigel. 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. AdHI-siRNA/VEGF can interfere tube formation.