Expression, purification and function of hVEGF_(121)/EGFP fusion protein in bone marrow-derived mesenchymal stem cells
10.3969/j.issn.1673-8225.2009.49.026
- VernacularTitle:骨髓间充质干细胞表达的hVEGF_(121)/EGFP融合蛋白的提纯及功能鉴定
- Author:
Lipeng HE
;
Yaosheng WANG
;
Yihua ZHOU
;
Yu JIANG
;
Xiaoling WU
;
Xiaoshu CHENG
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2009;13(49):9731-9734
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE: To purify hVEGF_(121)/EGFP fusion protein using transfected BMSCs as culture media, in addition, to detect the function of hVEGF_(121)/EGFP fusion protein in vitro.METHODS: The pEGFP-N_2-hVEGF_(121) recombinant plasmid, which was constructed in the preliminary work of our study group,was used to extract the plasmid DNA. BMSCs were transfected with pEGFP-N2-hVEGF_(121) by positive ionic liposome transfection method. Under a fluorescent microscopy, the expression of hVEGF_(121)/EGFP fusion protein was detected. The hVEGF_(121)/EGFP fusion protein was purified with Am icon ultrafiltration centrifuge tube and the expression of fusion protein was detected by Western-Blotting method.RESULTS: The BMSCs, which transfected with pEGFP-N2-hVEGF_(121), was observed under the fluorescent microscope. Western blotting confirmed that pEGFP-N_2-hVEGF_(121) fusion protein expressed in the culture media of transfected BMCS. MTT results showed the number of human umbilical vein endothelial cells in the fusion protein team was significantly greater than that in the control group (P < 0.05), and Miles test confirmed that pEGFP-N_2-hVEGF_(121) fusion protein increased the permeability of the blood vessel wall.CONCLUSION: ①This study successfully confirmed the pEGFP-N_2-hVEGF_(121) recombinant plasmid, which carrying VEGF_(121)/EGFP fusion protein, can be expressed in BMSCs.②The VEGF_(121)/EGFP fusion protein have the function of wild-type VEGF in vitro.
