| Objective1. Bone Mesenchymal Stem Cells (BMSCs) were isolated, cultured, amplified, purified in vitro to meet the large demand of seeded cells of bone engineering.2. To identified the multilineage differentiation potential and osteogenic differentiation capacity of BMSCs, osteogenic induction medium was used to cultured it and evaluate the osteogenesis.3. hBMP-2gene carried by pcDNA3.1plasmid was transfected into BMSCs mediated by Liposome, and the effects of transfection were observed.4. The influence of the transfection on the proliferation and differentiation of BMSCs was detected.Methods1. BMSCs’in-vitro cultivation:Limbs were extracted from newborn healthy New Zealand white rabbits at sterile condition after sacrificed. The complete medium was used to flush the bone marrow cavity, and then the solution was cultured by whole marrow adherent method. After6to7days’culture, primary cells could be digested for passage cultured at1:3and every passage cells could be cultured for2to3days.2. BMSCs’directional osteogetic differentiation:Cells of passage3were harvested for osteoanagenesis inducing for14to21days using osteoblast differentiation medium containing dexamethasone, β-sodium glycerophosphate, ascorbic acid. Mineralization nodules, ALP and BGP in the BMSCs after induction were detected by Alizarin Red staining, ELISA and IHC methods respectively.3. Liposome-mediated transfection of hBMP-2gene into BMSCs:The pcDNA3.1plasmids carrying liposome-mediated hBMP2gene were transfected into BMSCs of passage3. GFP was observed under fluorescence microscope. The instantaneous expression of hBMP-2mRNA was detected by RT-PCR.4. The proliferation ability of the tranfected cells was investigated by MTT method and flow cytometry respectively, while the differentiation capability of it was detected by IHC of BMP-2.Results1. Results of the in-vitro cultivation of BMSCs:6to7days after cultivation, primary BMSCs were overgrowthing for passage cultured. After subculturing, in addition to the preservation of typical morphological feature and growth state of BMSCs, under microscope, spindle-shaped cells and vortex arrangement were observed.2. Results of BMSCs’osteogenic differentiation:BMSCs could be osteogenicly induced successfully. After induction, positive dying of calcium nodules was observed. There was significant difference (p<0.05) in the contents of ALP between uninduced group and induced one in which the content reached highest on2-week consolidation. In addition, positive staining of BGP was observed by immunohistochemistry3. Results of the transfection of hBMP2gene into BMSCs mediated by liposome: The transfection efficiency of plasmid-carrying hBMP2gene was approximately30%. The fluorescence intensity was rather poor on post-transfection24h but became stronger after48h. And the gene expression of hBMP-2could be detected.4. The result of MTT revealed that the proliferation ability of the transfected BMSCs did not change significantly. And the result of IHC indicated that there was positive staining of BMP-2in the transfected cells.Conclusions1. Rabbit bone mesenchymal stem cells (BMSCs) could be cultivated, amplified and purified by the whole marrow adherent method which was convenient and practical.2. The BMSCs could be directionally induced to osteoblasts.3. Extragenous hBMP-2gene could be successfully transfected into rabbit BMSCs by the mean of liposome-mediated.4. The transfection of hBMP-2gene had no significant influence on the proliferation of BMSCs but make it differentiated into osteogetic. |
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