Construction of human VEGF165 gene eukaryotic expression plasmid and its effect on proliferation of vascular endothelial cells.
BACKGROUND
After organ transplantation, rapid repair of injured vascular endothelial cell(VEC) is a key to prevent graft chronic dysfunction besides control of immunological rejection. Many studies have confirmed that vascular endothelial growth factor 165(VEGF165) could accelerate the repair of VEC injury, decrease thrombosis and thrombotic occlusion, and inhibit hyperplasia of the intima. This study was designed to construct eukaryotic expression plasmid pBudCE4.1/VEGF165, and observe its effect on the proliferation of VEC.


METHODS
The VEGF165 gene cloned from human heart tissue by RT-PCR was cloned into eukaryotic expression plasmid pBudCE4.1. The recombinant expression plasmid pBudCE4.1/VEGF165 was identified by restriction enzyme ( Hind III and BamH I) digestion analysis, and was sequenced. The pBudCE4.1/VEGF165 was introduced into VEC through lipofection transfection. The VEGF165 mRNA expression by Northern blot and VEGF165 protein expression was detected by immunocytochemical staining. The effect of expression protein on VEC proliferation was detected by flow cytometry.


RESULTS
The RT-PCR product of the VEGF165 gene was about 576 bp. Sequencing analysis revealed that the sequence of the amplified VEGF165 gene was identical with that in GenBank. Restrictive enzyme digestion analysis showed that recombinant expression plasmid pBudCE4.1/tVEGF165 had been constructed successfully. The expression of VEGF165 at mRNA and protein levels in the transformed VSMCs had been demonstrated by Northern blot and immunocytochemical staining respectively. The expressed product of VEGF165 could notably accelerate the proliferation of VECs.


CONCLUSIONS
pBudCE4.1/VEGF165 is successfully constructed and is expressed in VECs. Expressed VEGF165 can accelerate the VEC proliferation. The present study has laid a foundation for potential use of VEGF165 gene transfection to prevent and treat vascular stenosis in the transplanted organ.
Wu
Zhong-Jun
ZJ
Organ Grafting Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China.
Zhang
Hong
H
Yu
Lin
L
Shi
De
D
Zheng
Shu-Sen
SS
D001483:Base Sequence; D015152:Blotting, Northern; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000166:cytology / Q000378:metabolism; D005784:Gene Amplification; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D032389:Myocytes, Smooth Muscle / Q000166:cytology; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D011995:Recombination, Genetic; D015183:Restriction Mapping; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000494:pharmacology*
D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A