Construction of recombinant adenovirus vector encoding VEGF siRNA and its inhibitory effect on K562 cell proliferation
- VernacularTitle:VEGF特异siRNA重组腺病毒构建及其对K562细胞增殖的抑制作用
- Author:
Fangfang LI
;
Youhua XU
;
Qing LUO
- Publication Type:Journal Article
- Keywords:
RNA interference;
cell proliferation;
vascular endothelial growth factor
- From:Journal of Third Military Medical University
2003;0(13):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct an recombinant adenovirus vector of vascular endothelial growth factor small interfering RNA(Ad5-VEGFsi) and to observe its inhibitory effect on the cell proliferation of human leukemia K562 cells. Methods The specific human VEGF siRNA was subcloned into the shuttle plasmid pSES-HUS and then cotransfected with plasmid pAdeasy-1 to produce pAd5-VEGFsi by homologous recombination. The identified recombinant plasmid pAd5-VEGFsi was packaged and amplified in 293 cells. At 72 h after the transfection of Ad5-VEGFsi into K562 cells, VEGF mRNA expression and the protein level of VEGF in the cell culture supernatant were determined by RT-PCR and ELISA, respectively. Cell proliferation was measured by MTT assay and cell apoptosis by flow cytometry. Results The recombinant adenovirus Ad5-VEGFsi was successfully constructed. Ad5-VEGFsi with the titer of 4.6?1011 pfu/ml was harvested by CsCl gradient purification. Compared with those in the control, EGF mRNA in cells decreased by 66.55% (P
