Construction of vascular endothelial growth factor modified muscle-derived stem cells and its expression
10.3969/j.issn.1006-5725.2015.18.005
- VernacularTitle:人血管内皮生长因子基因修饰的肌源性干细胞的构建及表达
- Author:
Geng AN
;
Xiangjin KANG
;
Wen ZHANG
;
Jinming ZHANG
- Publication Type:Journal Article
- Keywords:
Lentiviral vectors;
VEGF;
Muscle-derived stem cells;
Transfection
- From:
The Journal of Practical Medicine
2015;(18):2954-2956
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the lentiviral vector encoding vascular endothelial growth factor gene and detect the vascular endothelial growth factor (VEGF) expression in muscle-derived stem cells (MDSCs). Methods To culture MDSCs and detect the CD34,CD45,Bcl-2 and Desmin expression in MDSCs by immunofluorescence. A cDNA encoding VEGF gene was amplified by PCR. This fragment was cut with EcoRI and BamHI and ligated with an EcoRI- and BamHI-reated lentiviral vector pCDH-CMV-MCS-EF1-copGFP. Then DNA sequencing analysis was performed to confirm successful construction of pCDH-CMV-MCS-EF1-copGFP -VEGF. The expression of VEGF was confirmed using enzyme-linked immunosorbent assay (ELISA), Western blot, and Real-time PCR analyses. Results The pCDH-CMV-MCS-EF1-copGFP-VEGF lentiviral vector was constructed successfully. When MOI values in the transfection efficiency MDSCs by FCM. were 1,5,15, the transfection rate reached to 16.7%, 45.6%, 66.3% and 85.6% respectively. When MOI value was of 20, the rate was up to 90.1%. Real-time PCR, Western blot and ELISA showed stable expression of VEGF in MDSCs. Conclusion We successfully constructed lentiviral vector carrying the VEGF and stable expression in MDSCs.
