Osteogenic Differentiation Of Goat Bone Marrow Mesenchymal Stem Cells Induced By Adenovirus-mediated BMP-2/bFGF In Vitro

Background:It is undoubted that bone marrow mesenchymal stem cells (BMSCs) are the ideal seed cells for tissue engineering bone. A variety of cytokines are involved in the repair of bone injury, such as bone morphogenetic protein 2 (BMP-2) in transforming growth factor (TGF-β) superfamily, the most accurate member inducing bone formation, as well as basic fibroblast growth factor (bFGF) , a mitosis sources with broad spectrum, which can promote the formation of cartilage and bone tissue, but also directly stimulate angiogenesis to improve the new bone formation of implanted tissue engineering bone , and can also induce bone occurrence with BMP-2 and play an important role in bone healing process. The osteogenic activity of BMSCs induced by BMP-2 and bFGF together is greater than bFGF or BMP2 alone. However, exogenous bone induction factors easily lose activity through protease breakdown and are dispersed and diluted, with short continuous effect time and low efficiency, and can not play continuous local stimulation and bone induction effect.Therefore, gene therapy inducing bone formation through transgene can overcome the lack of direct application of exogenous growth factors.The choice of a suitable vector carrying the target gene is the key to gene therapy. Adenovirus is one of the most widely used gene vector efficiently into cells. It is a non-pathogenic replication-defective vector and can provide long-term gene expression with very low immunogenicity. It has wide host range and can infect humans, mice and other genera, with a range of organizations affinity. It exists as an additional body form after infecting host, and the genes are not integrated into the host cell genome, without the danger of insertion mutations activating oncogene. The foreign gene can be expressed freely for several weeks or months and disappears thereafter avoiding the continued bone proliferation caused by the over-expression of target gene, which meets the requirement of gene therapy for bone repair . The purpose of this study for construction and Identification of Recombinant Adenovirus Vector Co-expressing BMP-2 and bFGF genes,Osteogenic differentiation of goat bone marrow mesenchymal stem cells induced by adenovirus-mediated BMP-2/bFGF in vitro,etc, the experimental results activity indicating great potential for treating osteoporosis.Objective:1, To construct the recombinant adenovirus vector co-expressing BMP-2 and bFGF genes, and to verify the correct recombination.2, To investigate the gene expression and bone formation of goat bone marrow mesenchymal stem cells (BMSCs) transfected by adenovirus-mediated bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (bFGF).Methods:1, BMP-2 cDNA and bFGF cDNA were amplified by PCR, then connect the two ends of the Link series T2A, through the complementary T2A Peptide and extended the BMP2-T2A-bFGF to get the full-length PCR products finally, and insert them into the vector of pSuttle-IRES through two restriction points,which are BglII and EcoRV, cleavage and sequencing appraisal. Using the homologous recombination technology which can transfer expression cassettes of purpose fragment from the shuttle vector to adenovirus vector, and identificat positive clones. The PacI linearized adenovirus vector was transfected into 293A cells.Observed the expression of GFP; Extensive amplification after identified by PCR,and purified by CsCl gradient centrifugation; measured OD260, and calculated the virus titers.2, Heparinized bone marrow cwas collected from goat iliac crest under sterile conditions and adherent bone marrow mesenchymal stem cells (BMSCs) were cultured and isolated by density gradient centrifugation, then the second generation of isolated cells were transfected by Ad-GFP adenovirus (titer 1.9×1012VP/mL) MOI (multiplicity of infection) values: 0,1000,2000,3000,5000,7000,9000,11000] for 48 hours, then cells were observed under fluoroscope. An optimum MOI value was chosen to transfected 2 random groups of second generation of isolated BMSCs for further use. Reproduction curves of Ad-GFP & Ad-BMP2-bFGF-GFP adenovirus treated groups of BMSCs (titer 3.6×10¹¹VP/mL) as well as untreated BMSCs (control group) were produced with MTT method. Supernatant fluid were collected from Ad-BMP2-bFGF-GFP adenovirus group and untreated group respectively and stored at -80 0C for 18 days then dyed with alkaline phosphatase (ALP) activity staining kit. On the 22th day, expressions of BMP-2, bFGF were detected and compared with hBMP-2 ELISA kit,h-bFGF ELISA kit.Results:1, The BMP-2 and bFGF genes were verified by gene sequencing. Digested by Bglâ…¡and EcoRâ…¤to get the full-length sequences of BMP2-bFGF two genes ,were cloned into the shuttle plasmid and the adenovirus vector ,After two weeks we can observed significant expression of GFP accumulation, it indicates that the virus was packaged together successfully. The determination of titer was 3.6×10¹¹VP/ml.2, In Ad-GFP group, 83% cells were successfully transfected under inverted microscope when MOI was 5000. Reproduction curves showed neither Ad-GFP nor Ad-BMP2-bFGF-GFP induced significant influence on the growth of BMSCs. But some BMSCs of Ad-BMP2-bFGF-GFP treat group were in polygonal or cubical shapes indicating a tendency of becoming bone-formation, which was confirmed by ALP staining. ELISA detection showed significant differences in the expression of BMP-2 and bFGF between Ad-GFP and negative control group 48 hours after tarnfection. (t = 30.313, P< 0.01,t=49.977,P< 0.01), and the difference was most obvious 6 days after transfection, and can be detected during a period of about 20 days.Conclusion:1, we successfully constructed the co-expression vector of BMP2 and bFGF genes which is adenovirus vector. Lay a foundation for the further research of transfected human mesenchymal stem cells differentiate into osteoblasts.2, Ad-BMP2-bFGF-GFP-transfected BMSCs shows significant bone-formation tendency and high level of expression activity indicating great potential for treating osteoporosis.