[Effect of human VEGF165 in vitro on proliferation of vascular endothelial cells].
OBJECTIVE
To observe the effect of human VEGF165 on the proliferation of vascular endothelial cells(VECs) in vitro.


METHODS
VEGF165 gene was amplified by RT-PCR and 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 confirmed by DNA sequencing. The pBudCE4.1/VEGF165 was transfected into VECs through lipofection transfection. Expression of VEGF165 was detected by Northern blot and immunocytochemical staining. The effect of expressed VEGF165 on VEC proliferation was detected by flow cytometry.


RESULTS
Sequencing analysis revealed that the sequence of cloned VEGF165 gene was identical with that in GenBank (accession No.X62568). Restrictive enzyme digestion analysis showed that recombinant expression plasmid pBudCE4.1/tVEGF165 was constructed successfully. The expression of VEGF165 at mRNA and protein levels in the transfected VECs was demonstrated by Northern blot and immunocytochemical staining, respectively. The expressed VEGF165 notably stimulated the proliferation of VECs.


CONCLUSIONS
pBudCE4.1/VEGF165 is successfully constructed and expressed in VECs. Expressed VEGF165 can stimulate the VEC proliferation. The present study lays the foundation for using VEGF165 gene transfection to prevent and treat vascular stenosis in transplanted organ.
Wu
Zhong-jun
ZJ
The Transplantation Center, First Affiliated Hospital, Zhejiang University, Hangzhou 310003, China. wzjxdfpxy@zju.edu.cn
Wu
Wei-wei
WW
Yu
Lin
L
Shi
De
D
Zheng
Shu-sen
SS
D049109:Cell Proliferation; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D010957:Plasmids; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics
D012333:RNA, Messenger; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A