Osteogenesis of rabbit skin fibroblast transfected with core binding factor a1/osteoblast specific transplanting factor-2 gene.
- Author:
De-Chang XIAO
1
;
Lian-Fu DENG
;
Qing-Ming YANG
;
Wei ZHANG
;
Xue-Min LÜ
;
Wei FENG
Author Information
- Publication Type:Journal Article
- MeSH: Alkaline Phosphatase; biosynthesis; genetics; Animals; Cell Adhesion Molecules; biosynthesis; genetics; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; biosynthesis; genetics; Fibroblasts; cytology; physiology; Gene Expression; Genetic Vectors; Mice; Osteocalcin; biosynthesis; genetics; Osteogenesis; genetics; physiology; Rabbits; Reverse Transcriptase Polymerase Chain Reaction; Transfection
- From: Chinese Journal of Surgery 2005;43(4):247-251
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo 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).
METHODSCbfa1/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.
RESULTSCbfa 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.
CONCLUSIONSNew Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 can express osteogenesis-related genes and proteins, and form ossification nodules on their surface.