[Influence of liposome-mediated recombinant plasmid pIRES-hBMP-2-hVEGF165 on osteogenic activity of hBMSCs in vitro].
OBJECTIVE
To investigate the effects of the recombinant plasmid pIRES-hBMP-2-hVEGFl65 on differentiation and maturation of hBMSCs in vitro.


METHODS
The co-expressing vector of hBMP-2 and hVEGF165 was constructed. The BMSCs were isolated and cultured from health adult human denoted marrow. By the lipofection method, the reconstructed plasmids pIRES-hBMP-2-hVEGF165, pIRES-hBMP-2, pIRES-hVEGF165 and pIRES neo empty vector, were transfected to hBMSCs (groups A, B, C and D). The untransfected cells were harvested as control group (group E). After 4 weeks of culture, RT-PCR was employed to assay the hBMP-2, hVEGF165 and osteocalcin mRNA expression in hBMSCs. The expressions of hBMP-2 and hVEGF165 of BMSCs were assayed by Western blot. The level of ALP activities of BMSCs was determined. Col I was also determined by immunohistochemical staining.


RESULTS
Compared to group E, the hBMSCs in group A secreted high level of hBMP-2, hVEGF165, Col I and osteocalcin; osteocalcin and Col I expressed at high level in group B, and hVEGF165 expressed at high level in group C. Otherwise, the expression of hVEGF165 in group B and the expressions of hBMP-2 and Col I in group C resemble to that of groups D and E, no expression or few expression was observed. The activities of ALP in groups A, B, C, D and E were 0.91 +/- 0.03, 0.90 +/- 0.02, 0.64 +/- 0.03, 0.67 +/- 0.01 and 0.66 +/- 0.02, respectively. The activity of ALP of groups A and B were significantly increased compared with that of group E (P < 0.05); there was no significant difference among groups C, D and E (P > 0.05).


CONCLUSIONS
The recombinant plasmid pIRES-hBMP-2-hVEGF165 can be successfully transfected into BMSCs with cation liposome-mediated transfection method, the exogenous hBMP-2 and hVEGF165 genes can be expressed constitutively in the transfected BMSCs, and it can enhance the differentiation abilities of BMSCs.
Wu
Guoping
G
Department of Plastic and Burns Surgery, the Affiliated Hospital of Luzhou Medical College, Luzhou Sichuan, PR China. drgpwu@yahoo.com.cn
He
Xiaochuan
X
Yang
Zhihui
Z
Guo
Li
L
D000328:Adult; D001854:Bone Marrow Cells / Q000166:cytology; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics*; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D017886:Durapatite; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D008081:Liposomes / Q000378:metabolism; D010012:Osteogenesis / Q000235:genetics*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
D055396:Bone Morphogenetic Protein 2; D008081:Liposomes; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D017886:Durapatite