[Comparative study on osteogenic effect of bone marrow mesenchymal stem cells transfected by adenovirus-bone morphogenetic protein 2-internal ribosome entry site-hypoxia inducible factor 1alpha(mu) and by bone morphogenetic protein 2 single gene].
OBJECTIVE
To compare the osteogenic effect of bone marrow mesenchymal stem cells (BMSCs) transfected by adenovirus-bone morphogenetic protein 2-internal ribosome entry site-hypoxia inducible factor 1alpha(mu) (Ad-BMP-2-IRES-HIF-1alpha(mu)) and by Ad-cytomegalovirus (CMV)-BMP-2-IRES-human renilla reniformis green fluorescent protein 1 (hrGFP-1) single gene so as to optimize the source of osteoblasts.


METHODS
BMSCs were separated and cultured from 1-month-old New Zealand white rabbit. The BMSCs at passage 3 were transfected by virus. The experiment was divided into 4 groups (groups A, B, C, and D) according to different virus: BMSCs were transfected by Ad-BMP-2-IRES-HIF-1alpha(mu) in group A, by Ad-CMV-BMP-2-IRES-hrGFP-1 in group B, by Ad-CMV-IRES-hrGFP-1 in group C, and BMSCs were not transfected in group D. The optimum multiplicity of infection (MOI) (50, 100, 150, and 200) was calculated and then the cells were transfected by the optimum MOI, respectively. The expression of BMP-2 gene was detected by immunohistochemistry staining after transfected, the expressions of BMP-2 protein and HIF-1alpha protein were detected by Western blot method. The osteogenic differentiation potential was detected by alkaline phosphatase (ALP) activity and Alizarin red staining.


RESULTS
The optimum MOI of groups A, B, and C was 200, 150, and 100, respectively. The expression of BMP-2 was positive in groups A and B, and was negative in groups C and D by immunohistochemistry staining; the number of positive cells in group A was more than that in group B (P < 0.05). The expression of BMP-2 protein in groups A and B was significantly higher than that in groups C and D (P < 0.05), group A was higher than group B (P < 0.05). The expression of HIF-1alpha protein in group A was significantly higher than those in the other 3 groups (P < 0.05), no significant difference was found among the other 3 groups (P > 0.05). ALP activity in groups A and B was significantly higher than that in groups C and D (P < 0.05), group A was higher than group B (P < 0.05). Calcium nodules could be seen in groups A and B, but not in groups C and D; the number of calcium nodules in group A was higher than that in group B (P < 0.05).


CONCLUSIONS
The expression of BMP-2 and osteogenic effect of BMSCs transfected by Ad-BMP-2-IRES-HIF-1alpha(mu) (double genes in single carrier) are higher than those of BMSCs transfected by Ad-CMV-BMP-2-IRES-hrGFP-1 (one gene in single carrier).
Department of Orthopaedic and Joint Surgery, First Affiliated Hospital of Liaoning Medical College, Jinzhou Liaoning 121001, PR China.
Jieyuan
J
Zhang
Hong
H
Yuan
Chen
C
Li
Quanying
Q
Li
Wei
W
Guo
Danping
D
Liu
D000256:Adenoviridae / Q000235:genetics; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D005822:Genetic Vectors; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000378:metabolism; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects; D010012:Osteogenesis / Q000187:drug effects* / Q000235:genetics; D011817:Rabbits; D023822:Tissue Engineering / Q000379:methods; D014162:Transfection
D055396:Bone Morphogenetic Protein 2; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D000469:Alkaline Phosphatase