[Osteogenesis of rabbit skin fibroblast transfected with core binding factor a1/osteoblast specific transplanting factor-2 gene].
OBJECTIVE
To study osteoblastic phenotype expression of New Zealand rabbit skin fibroblasts transfected with mouse core binding factor a1/osteoblast specific transplanting factor-2 gene (Cbfa1/Osf2).


METHODS
Cbfa1/Osf2 gene, engineered into eukaryotic expression vector pSG5, was introduced into New Zealand rabbit skin fibroblasts with catholyte liposomes-Lipofectamine 2000. Meanwhile, those transfected pSG5 and un-transfected were set the control groups. The expression of Cbfa1 gene, osteocalcin (OCN) gene, alkaline phosphatase (ALP) gene and pre-peptide 2 alpha gene of collagen type I were detected by RT-PCR assay. Cbfa1 protein was detected by Western-Blot assay, in-cell ALP activity by p-nitrophenyl phosphate (PNPP) assay and OCN content in the supernatant by radio-immunity method. The ossification nodules was detected by Alizarin-Red staining and scanning electron microscope.


RESULTS
Cbfa 1mRNA and Cbfa1 protein were expressed in New Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 from the first day to the fifth day, but they were not detected in the control groups. In the pSG5-Cbfa1/Osf2 transfected group, the expression of ALP gene and OCN gene were respectively induced from the third day and the forth day, pre-peptide 2 alpha gene of collagen type I was enhanced from the third day. From the sixth day, ALP activity greatly increased, OCN strongly secreted, and they were maintained at a high level for about 4 weeks, and the difference was significant compared with the control group (P < 0.05). On the forty-second day, ossification nodules were found on the surface of pSG5-Cbfa1/Osf2 gene transfected cells.


CONCLUSIONS
New Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 can express osteogenesis-related genes and proteins, and form ossification nodules on their surface.
Xiao
De-Chang
DC
Department of Orthopaedic, Ruijin Hospital, Shanghai Second University, Shanghai 200025, China.
Deng
Lian-Fu
LF
Yang
Qing-Ming
QM
Zhang
Wei
W
Lü
Xue-Min
XM
Feng
Wei
W
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D015815:Cell Adhesion Molecules / Q000096:biosynthesis / Q000235:genetics*; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000096:biosynthesis / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D015870:Gene Expression; D005822:Genetic Vectors; D051379:Mice; D015675:Osteocalcin / Q000096:biosynthesis / Q000235:genetics; D010012:Osteogenesis / Q000235:genetics* / Q000502:physiology; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D015815:Cell Adhesion Molecules; D050659:Core Binding Factor Alpha 1 Subunit; C082838:Postn protein, mouse; D015675:Osteocalcin; D000469:Alkaline Phosphatase