Detection of protein secretion and expression and osteogenic activity of adipose tissue-derived stem cells after in vitro transfection of vascular endothelial growth factor

Abstract

To examine vascular endothelial growth factor (VEGF) protein secretion and expression and explore the osteogenic activity of adipose tissue-derived stem cells (ADSCs) after transfection of human VEGF. The ADSCs were isolated from human adipose tissue after the digestion of collagenase.After identification by flow cytometry, the cells were cultured and passaged in nutritive medium. Gene sequence encoding human VEGF mature peptide was obtained by Trizol reagent method from human vascular tissue. Target gene VEGF was connected with bicistronic expression vector containing green fluorescent protein to form pSELECT-GFP zeo-VEGF for transfecting 2nd, 3rd, 4th, 5th generation ADSCs mediated by liposome. The transfection results were verified under fluorescence microscope. VEGF protein secretion by transfected cells was detected by enzyme-linked immunosorbent assay (ELISA). Second-generation transfected ADSCs were cultured under osteogenic conditions.The supernatant levels of alkaline phosphatase (ALP) and osteocalcin (OC) were detected. Liposome-mediated VEGF target gene fragment could transfect ADSCs successfully. ELISA quantitative detection showed that VEGF mRNA expression levels in supernatant of the transfected group was significantly higher than the control group. And there were significant differences. After osteogenic culturing, the detections of ELISA, real-time PCR and Western blot showed that the secretion of ALP and OC of VEGF transfected group was significantly higher than that of empty vector transfected and blank cell groups.And there were significant differences (P < 0.01). After transfected by liposome-mediated VEGF target gene fragment, human ADSCs can express biologically active VEGF mRNA in vitro continuously and effectively. Directional differentiation capacity of transfected ADSCs is significantly enhanced.