The Parameter Optimization Of Adenovirus-mediated BMP-2 Gene Transfected Bone Marrow Mesenchymal Stem Cells

Background: Bone marrow mesenchymal stem cells(BMSCs)is within the bone marrow have multilineage differentiation potential of a kind of non hematopoietic stem cells,under appropriate microenvironment,differentiation into bone cells,cartilage cells,fat cells,muscle cells,and so on.It is the ideal seed cells of gene engineering and tissue engineering,and to be the hot research direction of the bone(cartilage)defect repair.However,bone morphogenetic protein(BMP)family is a typical acidic glycoprotein,in the bone remodeling process,many factors plays an important regulatory role,especially BMP-2,can induce the formation of bone and cartilage,and can bind with a variety of carrier.In this study,by adenovirus vector mediated guide BMSCs transfected by BMP-2 gene,and then observe the morphology and osteogenic ability of mesenchymal stem cell,to lay a solid theoretical foundation for the construction of the experimental study on the animal model of bone defect and repair the defect.Objective: Discussion to experimental study of adenovirus mediated BMP-2 gene transfected BMSCs to observe and analyze the changes of gene and protein expression,and detection of BMP-2 factor on the vitality of the stem cells and the ability of bone changes,thus obtains the suitable conditions in transfection.Methods: Selected aged F344 rats,through extraction and separation of bone marrow mesenchymal stem cells,purification,amplification in vitro,and use adenovirus mediated BMP-2 gene transfect BMSCs,by ordinary optical microscopy and fluorescence microscopy observing changes in cell morphology and fluorescence,transfection on the proliferation and differentiation of BMSCs intervention changes were observed.Detecting the BMP-2 expression and changs in cell culture supernatant after transfection by Western blot,and by alkaline phosphatase assay and alizarin red staining experiments were used to detect the ALP content changes and calcium salt deposition nodules.From these to know the effects of BMP-2 on osteogenic differentiation and osteogenic induction of BMSCs.After BMP-2 gene was successfully transfected into BMSCs gene,the protein expression was positive,which could effectively induce the differentiation of BMSCs.We by quantifying the transfection conditions,the adenovirus infection(MOI)packet including 0,50,100,200,300,400MOI;cell seeding density culture includes 5000,10000,20000,40000cells/cm~2,to monitor the relative optimal transfection conditions and cell culture density.Results: From F344 rat bone marrow isolate and culture the bone marrow mesenchymal stem cells,cell morphology showed spindle shaped,fish shaped and spiral arrangement growth,in line with the characteristics that bone marrow mesenchymal stem cells had;Western blot showed BMSCs after gene transfection,the expression of BMP-2 is sustainable and high.In vitro,the BMP-2 could significantly promote the proliferation and differentiation of BMSCs;through quantitative detection of alkaline phosphatase showed BMSCs transfected with higher ALP expression;the alizarin red staining showed BMSCs gradually nodules of calcium salt deposition after BMP-2 gene transfected,the content reached the peak when transfected about 21 days,staining well.Conclusions:(1)After adenovirus mediated BMP-2 gene transfected BMSCs,cells can be continued to maintain their stem cell activity and proliferation and differentiation.BMSCs can produce BMP-2 successfully,so that the osteogenic activity of stem cells is obviously enhanced.(2)The appropriate conditions of transfection: multiplicity of infection is 100 cells,planting density is 20000 cells/cm~2,the BMSCs can express BMP-2 protein stablely and lastingly,promote cells into bone more effectively and osteogenic activity reached its maximum.