Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (24): 4427-4431.doi: 10.3969/j.issn.1673-8225.2011.24.014

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Construction of basic fibroblast growth factor gene fluorescent eukaryotic cell expression vector and inhibitory effect on hydrogen peroxide-induced apoptosis and related gene expression in vascular endothelial cells

Xu Bin1, Lin Gui-xian2, Wu Xiao-ying2, Mao Jian-wen2   

  1. 1School of Life Science and Biopharmacology, 2School of Basic Medicine, Guangdong Pharmaceutical University, Guangzhou  510006, Guangdong Province, China
  • Received:2010-12-29 Revised:2011-03-19 Online:2011-06-11 Published:2011-06-11
  • Contact: Mao Jian-wen, Associate professor, School of Basic Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, Guangdong Province, China jianwenmao@ hotmail.com
  • About author:Xu Bin☆, Doctor, Associate professor, School of Life Science and Biopharmacology, Guangdong Pharmaceutical University, Guangzhou 510006, Guangdong Province, China xubin@gdpu.edu.cn
  • Supported by:

    Startup Fund for Doctor of Guangdong Pharmaceutical University, No. 2006SMK03* 

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Abstract:

BACKGROUND: It has been verified that exogenous basic fibroblast growth factor (bFGF) has obvious inhibitory effect on apoptosis in vascular endothelial cells.
OBJECTIVE: To construct a fluorescent eukaryotic cell expression vector carrying the gene of human bFGF, and to investigate its effect on apoptosis induced by hydrogen peroxide (H2O2) and related gene expression in vascular endothelial cells.
METHODS: bFGF and GFP gene was subcloned into eukaryotic expression plasmid pcDNA3.1 by means of gene cloning to obtain pcDNA3.1-bFGF-GFP. pcDNA3.1-bFGF-GFP was transfected into umbilical vein endothelial cells by liposome mediating, RT-PCR and observing green fluorescence by fluorescence microscope were used to determine the expression of bFGF and GFP. Umbilical vein endothelial cells were divided into three groups: control group (transfected pcDNA3.1), H2O2 group (transfected pcDNA3.1+H2O2) and bFGF-transfected+H2O2 group (transfected pcDNA3.1-bFGF-GFP+H2O2), the apoptosis of umbilical vein endothelial cells was detected by flow cytometry, the expression of caspase-3 P17 subunit and bax protein were determined by Western blot.
RESULTS AND CONCLUSION: pcDNA3.1-bFGF-GFP was successfully constructed, bFGF mRNA was increased significantly and specific green fluorescence was observed by fluorescence microscope after pcDNA3.1-bFGF-GFP transfected. Compared with the control group, apoptosis rate, the expression level of caspase-3 P17 subunit and bax protein significantly increased in H2O2 group (P < 0.01). Compared with H2O2 group, bFGF gene transferring significantly decreased apoptosis rate, down-regulated the expression of caspase-3 P17 subunit and bax protein (P < 0.01). bFGF gene transferring can inhibit the apoptosis induced by H2O2 in vascular endothelial cells, the underlying mechanism might be associated with regulation on the expression of bax protein and activity of caspase-3.

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