Study Of BMP2and EGFP Recombinant Adenovirus Transfection On Rabbit Bone Marrow Derived Mesenchymalstem Cells In Vitro

Objective1. In order to establish an effective BMSCs cultured approachin vitro in this experiment.2. Leading human bone morphogenetic protein-2(hBMP-2) and EGFP gene transfected rabbit BMSCs by adenovirus medium,through line Westenblot and immunocytochemistry to observe the expression ofBMP2and EGFP. Methods1. The bone marrow liquid was extracted at thefemoral of the rabbit by medullo-puncture needle. The BMSCs was separatedand cultured by density gradient centrifugation and adherence method andobserved in vitro.2. BMSCs was induced by osteogenesis induced fluid(containing dexamethasone10-8mol/L, vitamin C50mg/L,β-glycerophosphate10mmol/L, the volume fraction of15%fetal bovine serumL-DMEM culture medium), collagen typeⅠ, alkaline phosphates (ALP),calcium nodules were respectively detected at the7th day，14th day，and afterthe induction detection calcium nodules on the21th day; Detecting the marks onthe surface of cells and period by flow cytometer.3. Detecting the expression ofexogenous gene in the cells by Immunocytochemical stain and Westenblot.Results1. A large amount of active BMSCs can be isolated by density gradient centrifugation and adherence method. Observed them48hours afterinoculation，the cells grew in spindle、swirling and radial shape. Generally about4~5d bottom wall of the culture bottle can be full covered.2. About56.84%ofthe rabbit BMSCs cells’ period is in G1phase; CD44shows positive, CD45shows negative; induced by the osteoblast, collagen type Iimmunohistochemical staining showed cytoplasmic brown, while normalcytoplasm were unstained. ALP enzyme linked immunosorbent assay showedthat compared with the normal group, the content of the induction group wassignificantly increased. Induction group Alizarin red staining was positive, butthe normal group was negative.3. After transfection, collagen typeⅠ wasactive in the induced group, in normal group typeⅠ collagen was negative.Respectively extracted48h after transfection and not transfection cell protein,Western-blot detected Ad-BMP2/EGFP group had positive bands, and Ad-EGFPgroup and the non-transfected group stripe is weak. The BMP2aim proteins incells after transfection rabbit BMSCs s highly expressed. Conclusion1.Densitygradient centrifugation and adherence method can isolate higher purity andactivity of BMSCs.2. After gene transfecting bone marrow stromal stem cells,it confirms that the transfecting cells can efficiently express BMP-2and promotetheir proliferation and differentiation by immunohistochemical and Westenblotexperiments.