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Construction and angiogenic effects of recombinant adenovtral vector expressing human angiopoietin 1

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Author:
No author available
Journal Title:
Chinese Journal of Biomedical Engineering
Issue:
4
DOI:
10.3760/cma.j.issn.1674-1927.2011.04.009
Key Word:
血管生成素1;腺病毒;基因载体;构建;血管形成;Angiopoietin-1;Adenovirus;Gene vector;Construction;Angiogenesis

Abstract: Objective To construct a recombinant adenoviral vector carrying and expressing human angiopoietin 1 (Ang-1) (Ad-Ang-1), and to examine the expression of Ang-1 in Ad-Ang-1-infected QBI-293A target cells and the angiogenic effects of Ang- 1. Methods The Ang- 1 fragments were amplified by RT-PCR using human marrow cells total RNA as templates and subcloned into pAdTrack-CMV transfer vector at Bgl Ⅱ and Sal Ⅰ sites to construct the recombinant transfer plasmid pAdTrack-CMV-Ang-1. The pAdTrack-CMV-Ang-1 transfer vector and pAdEasy-1 backbone vector were further cotransformed into the bacteria BJ5183 cells for homologous recombination. The pAdEasy-1-pAdTrack-CMV-Ang-1 (pAd-Ang-1) homologous recombinant plasmids were transfected into the human embryonic kidney 293 (QBI-293A) cells, which were then amplified and purified,resulting in high-titer recombinant adenovirus Ad-Ang-1. Adenovims-mediated transcription and expression of exogenous Ang-1 gene in QBI-293A cells were examined by RT-PCR and ELISA, respectively. Chick embryos were randomized into 3 groups: Ad-group, Ad-Ang-1 group and normal saline (NS) group. The angiogenesis on chick chorioallantoic membrane was examined. Results High-titer Ad-Ang-1[(3×1010) pfu/ml]adenovirus was obtained. Compared with QBI-293A cell control[(2490.86±550.95 ) ng/L]and Ad-infected QBI-293A blank adenovirus control group[(2 323.20±260.55) ng/L], Ad-Ang-1-infected QBI-293A cells significantly transcribed and expressed higher levels of Ang- 1[(47522.53±3685.57 ) ng/L](P<0.01). Compared with the Ad group and NS group, the Ad-Ang- 1 group significantly promoted angiogenesis with formation of second and third generation vessels. Conclusion The Ad-Ang- 1 adenoviral vector expressing Ang- 1 is successfully constructed, and AdAng-1 recombinant adenovirus may obviously promote angiogenesis on the chick chorioallantoic membrane, which provides the experimental evidence for further research on Ang-1 gene-modified stem cells.

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