The Research On The Sheep Bone Marrow Mesenchymal Stem Cells Transfected By BFGF Gene And Its Regulation Of Osteogenic Potential

Objective: To observe the effect of whole bone marrow culture and Percoll densitygradient centrifugation on biological activity of sheep bone marrow mesenchymal stemcells. And to induce BMSCs which is better biological activity to differentiate intoosteoblasts, and to understand gene kinetic expression patterns during the bone marrowstromal cells to differentiate process. Methods: To obtain BMSCs by the whole bonemarrow culture and density gradient centrifugation, and to identify the CD44antigenexpression by flow cytometric. The potential of osteogenic differentiation is examinedutilizing Von Kossa’s staining and alkaline phosphatase staining. Better culture methodis adopted to extract sheep bone marrow mesenchymal stem cells, and using commonculture medium and mineralization induction medium for BMSCs culture. With real-timePCR tests the quantity change of osteogenesis gene expression of two groups of bonemarrow mesenchymal stem cells. To construct lentiviral vector carrying human bFGFgene and GFP gene. bFGF-lentivirus and GFP-lentivirus supernatant was collected andinfected the fourth generation bone marrow mesenchymal stem cells. And Flowcytometric detects transfection efficiency. With real-time PCR tests the quantity changeof osteogenesis gene expression of two groups bone marrow mesenchymal stem cellswhich are transfected and not transfected. Results: CD44positive rate of BMSCs ofwhole bone marrow culture is87.6%, and Percoll density gradient centrifugation’s is83.4%, the difference of CD44positive was not statistically significant(P>0.05). VonKossa’s method mineralization nodules dyeing and staining of alkaline phosphatase arepositive. To compare with non-induction group, the expression of osteocalcin andtypeⅠcollagen were enhanced at second, third and fourth passengs of cells(P<0.05), andthe expression of osteopontin at cells of the first, second, third and fourth generations are not different(P>0.05). In induced BMSCs, the maximum expression of typeⅠ collagenappear at second generation cells(P<0.05), and the maximum expression of osteocalcinappear at the fourth generation cells(P<0.05), and the expression of osteopontin at cellsof the first、second、third and fourth passengs were not different(P>0.05).96h aftertransfection, GFP-lentivirus transfect bone marrow of mesenchymal stem cells efficiencyis87.2%. To compare with non-transfection group, OPN gene expresses high quantity,but no difference (P>0.05), Collagen-Ⅰe xpresses low quantity(P<0.05) and OCexpresses high quantity (P<0.05), the differences are statistically significant. Conclusion:The whole marrow culture is an effective method of BMSCs. These sheep BMSCsgradually differentiated into osteoblasts following in vitro mineralization. The forthgeneration of induction culture of bone marrow mesenchymal stem cells has had thecharacteristics of osteoblasts and osteogenetic ability. The change of osteogenic genesexpression in the transfected groups are more close to the osteoblast mineralization atmaturity. These cells can be used as the seeds of bone tissue engineering cells for furtherstudy.