In vitro expression and in vivo osteogenic capability of pcDNA3-hBMP2-transfected marrow stromal cells in rabbits / 中国组织工程研究
Chinese Journal of Tissue Engineering Research
;
(53): 7587-7590, 2008.
Article
in Chinese
| WPRIM
| ID: wpr-406947
ABSTRACT
BACKGROUND:
Whether bone morphogenetic protein 2 (BMP-2) can be transduced into marrow stromal cells (MSCs) and produce osteogenic effects by viral or non-viral vector remains unclear?OBJECTIVE:
To observe the expression of cultured rabbit MSCs transfected with pcDNA3-hBMP2 in vitro. Simultaneously, the MSCs were transfected but not screened and then transplanted into autologous muscle to investigate the osteogenic capability by X-ray. DESIGN, TIME ANDSETTING:
A controlled observation experiment was performed at the Department of Orthopedics, Liaoning Medical University between November 2004 and April 2005. MATERIALS Six adult New Zealand rabbits, of either gender, weighing 2.0-3.0 kg, were included for this study. BMP2 antibody was the product of Sanaka Company, USA. pcDNA3-hBMP2 was provided by Professor Pu Qin from the Department of Biochemistry, Fourth Military Medical University of Chinese PLA). Restriction enzyme was purchased from Takara biotechnology (Dalian) CO., LTD., China.METHODS:
Super-purified plasmid pcDNA3-hBMP2 was extracted from E. coli. Bone marrow was taken from the adult rabbit femur for harvesting MSCs by density gradient separation. The MSCs were divided into the following 4 groups Group A, cells were transfected and screened by G418; Group B, cells were transfected by pcDNA3-hBMP2; Group C, cells were transfected by empty vector pcDNA3; Group D, only transfection reagent Fugene 6 was added.MAIN OUTCOMEMEASURES:
Transient BMP2 expression was analyzed by immunohistochemistry. Expression of osteocalcin and collagen I was examined by immunohistochemistry and in situ hybridization, respectively. Two weeks after transfection, MSCs from the group B were autologously transplanted into the muscle. Four weeks later, X-ray assay was used to observe bone formation.RESULTS:
pcDNA3-hBMP2 was successfully transduced into MSCs and transiently expressed BMP2 100%. Four weeks after gene transfection, expression levels of osteocalcin and collagen I were significantly higher in the group A than in the groups C and D. X-ray results demonstrated new bone formation four weeks after MSCs transplanted into the muscle.CONCLUSION:
pcDNA3-hBMP2 can safely and efficiently transfect MSCs and induce them to differentiate towards osteoblasts by secreting BMP2.
Full text:
Available
Index:
WPRIM (Western Pacific)
Language:
Chinese
Journal:
Chinese Journal of Tissue Engineering Research
Year:
2008
Type:
Article
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