[Transfection of human vascular endothelial growth factor 165 gene into rat bone marrow mesenchymal stem cells in vitro].
OBJECTIVE
To construct a recombinant adenovirus vector containing human vascular endothelial growth factor 165 (hVEGF165) [pAdxsi-enhanced green fluorescent protein (EGFP)-hVEGF165], and to observe the expression of hVEGF165 by transfecting pAdxsi-EGFP-hVEGF165 into rat bone marrow mesenchymal stem cells (BMSCs) in vitro so as to lay a foundation for further research on gene therapy of blood vessel regeneration.


METHODS
hVEGF165 was liberated from plasmid and was subcloned into pShuttle-EGFP. The pShuttle-cytomegalo-virus-EGFP was then transferred to pAdxsi vector, by which pAdxsi-EGFP-hVEGF165 virus plasmid was obtained and was identified by enzymes restriction analysis and gene sequencing. The pAdxsi-EGFP-hVEGF165 was linearized by digestion with restriction endonuclease PacI, and was then transfected into human embryonic kidney cells (HEK293). The retrieved recombinant adenovirus was titrated by using 50% tissue culture infective dose assay. The rat BMSCs were cultured and were infected with recombinant adenovirus containing EGFP (pAdxsi-EGFP). The multiplicities of infection (MOI) of transfection were determined by fluorescent inverted phase contrast microscope and flow cytometry (FCM), by which the most optimal value of MOI was confirmed and was used for transfecting pAdxsi-EGFP-hVEGF165 into BMSCs. The expression of hVEGF165 gene was identified by performing Western blot, RT-PCR, and ELISA. The effect of transfection on BMSCs proliferation was assessed by MTT.


RESULTS
The expression of hVEGF165 cDNA in recombinant adenovirus plasmid was identified by enzymes restriction analysis and gene sequencing. The titer of virus could be up to 1 x 10(10) pfu/mL after several rounds of transfection and amplification. The efficiency of transfection on FCM was 88% when MOI being 150 pfu/cell, at which the most optimal of MOI was achieved, as observed on fluorescence. The expressions of hVEGF165 at both mRNA and protein levels were detected after 48 hours of the transfection. The results of ELISA showed the expression of hVEGF165 peaked at 7 days, and the production was found even after 20 days. Furthermore, the expression of hVEGF165 protein at 1, 3, 5, 7, 9, 11, 13, 15, and 20 days in the group transfected with pAdxsi-EGFP-hVEGF165 was significantly higher than that in the group transfected with pAdxsi-EGFP and in untransfected group (P < 0.05). The results of MTT demonstrated that there was no significant difference in absorbance (A) value between transfected with pAdxsi-EGFP-hVEGF165 group and untransfected group (P > 0.05).


CONCLUSIONS
BMSCs are suitable for gene transfection, and hVEGF165 gene can be transferred into BMSCs with high efficiency using pAdxsi-EGFP-hVEGF165 at a MOI of 150 pfu/cell. The transfected BMSCs can highly express hVEGF165, which has no effect on BMSCs growth and proliferation.
Department of Burns and Plastic Surgery, Lanzhou General Hospital, Lanzhou Military Command of Chinese PLA, Lanzhou Gansu 730050, PR China.
Wenjia
W
Liang
Qian
Q
Cai
Yi
Y
Liu
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002478:Cells, Cultured; D005822:Genetic Vectors; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D010957:Plasmids; D051381:Rats; D017208:Rats, Wistar; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A