[Differentiation of rabbit bone marrow mesenchymal stem cells into myogenic cells in vitro and expression of vascular endothelial growth factor gene after transfection].
OBJECTIVE
To induce the differentiation of rabbit bone marrow mesenchymal stem cells (MSCs) into myogenic cells in vitro, and to investigate the expression of vascular endothelial growth factor (VEGF) gene in AdTrackCMV-hVEGF165- transfected MSCs.


METHODS
Twenty rabbits were divided equally into control group and experimental group, and MSCs were isolated and purified from their bone marrow by Percoll (1.073 g/ml) followed by cell culture in low-glucose DMEM supplemented with 10% fetal bovine serum. 5-azacytidine (5-Aza) was added into the cell culture of the experimental group on the third day. The expression of troponin I in MSCs was assayed by immunohistochemistry on the 28th day. AdTrackCMV- hVEGF165 eukaryotic expression vector was constructed and transfected into the MSCs, and subsequent VEGF expression was detected by Northern blotting and Western blotting while enzyme-linked immunosorbent assay (ILISA) was employed to examine the VEGF concentration in the supernatant of the culture medium.


RESULTS
Following successful isolation and culture of the MSCs from rabbit bone marrow, 5-Aza-induced differentiation of the cells into myogenic cells was demonstrated by their positive staining for cardiac troponin I (cTnI). Northern blotting showed that the expression of VEGF 165 mRNA was much higher in the VEGF165 gene-transfected cells than in the control cells. Western blotting showed VEGF expression in the transfected cells. The concentration of VEGF in the supernatant mounted to the peak level 3-5 d after VEGF165 gene transfection (1,011-1,027 pg/ml) and decreased gradually thereafter, but still maintaining higher levels than those in the control group and pAdTrackCMV group (349 pg/ml vs 116 pg/ml and 125pg/ml, respectively, P<0.01).


CONCLUSIONS
MSCs can be induced to differentiate into myogenic cells in vitro and express VEGF after VEGF gene transfection, and this success may provided a basis for combining MSC transplantation with gene therapy for regeneration of the damaged myocardial cells.
Sheng
Xiao-gang
XG
Department of Cardiology, Guangdong Provincial Cardiovascular Institute, Guangzhou 510100, China. shengxiaogang1@sina.com.cn
Feng
Jian-zhang
JZ
Wu
Shulin
S
Jin
Li-jun
LJ
Yu
Xi-yong
XY
Zhang
Bin
B
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism; D002454:Cell Differentiation; D002455:Cell Division; D005260:Female; D007150:Immunohistochemistry; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000196:embryology*; D010957:Plasmids; D011817:Rabbits; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis / Q000235:genetics*
D042461:Vascular Endothelial Growth Factor A