Research On The Biological Properties Of Bone Marrow Mesenchymal Stem Cells Modified With HTGF-β1 And HBMP-2

Objectives:(1) To apply transgenic technology to modificate BMSCs with hTGF-β1 on the basis of the fact that hTGF-βinducing differentiation of BMSCs to osseous tissue.(2)To evaluate BMSCs ossification by virtue of the synergetic effect of hTGF-β1 and hBMP-2 and elucidate its value to be seed cells for osteogenic tissue engineering.Methods:1.The hTGF-β1 recombinant adenovirus and control adenovirus pAdMax-EGFP-3FLAG were constructed using the pAdMax system2. The establishment of bone mesenchymal stem cells modified by target gene(1) Rat MSCs were isolated and cultured by united means of differential adhesion and flow cytometry method in DMEM medium supplemented with 10% fetal bovine serum(2) The hTGF-β1 adenovirus were transfected into BMSCs and the appropriate MOI was verified by observing EGFP expression with fluorescent microscope(3) The mRNA expression of hTGF-β1 gene in BMSCs was detected by RT-PCR;ELISA was used to analyze the secretion of target protein3. The study of bioactivity of BMSCs modified with different geneWe divided those cells into 5 groups:A.BMSCs;B. BMSCs modified with EGFP;C. BMSCs modified with hBMP-2;D. BMSCs modified with hTGF-β1;E. BMSCs co-modified with hTGF-β1and hBMP-2.(1) MTT method was used to analyze the cell Proliferation of the different groups(2) Alkaline phosphatase (ALP) activity in endochylema was tested by enzymatic reactionResults:(1) hTGF-β1 gene were cloned into pAV-MCMV-GFP-3FLAG and the Ad-TGF-β1 were constructed;The results of DNA sequencing showed that the sequences we have cloned were identical with those in GeneBank.(2)Rat MSCs was successfully isolated,purified and expanded efficiently by the differential adhesion and flow cytometry method,while the eGFP-expression showed the target gene transfected into BMSCs in vitro.RT-PCR showed the mass transcription of hTGF-β1 in transfected BMSCs.ELISA confirmed the expression of hTGF-β1 gene in transfected cells and cell culture medium.(3)BMSCs got an increased proliferation ability after to have been transfected with different genes,especially the modification with hTGF-β1 and hBMP-2;The in vitro experiment results showed that the alkaline phosphatase(ALP) activity of BMSCs can be improved by hTGF-β1 and hBMP-2 gene co-modification.Conclusion:(1)A stable and effective co-expression of both genes can be achieved in transfected BMSCs through new AdMax adenovirus vector;(2)Combined use of hTGF-β1 and hBMP-2 can produce better osteogenesis than each single alone in virtue of the synergistic effect of both genes.