| Bone morphogenetic proteins (BMPs) have been widely studiedbecause of its diverse biological function since they were found. BMPsplay important roles in embryonic generation and development, cellulardifferentiation and the development of organs. Nevertheless, moreresearchers are more interested in the biological Osteoinductive activity ofBMPs for some multipotential stem cells. BMPs belong to thetransformation growth factor β (TGF-β) superfamily, and consist of at least40members (identification and separation about twenty members). BMP-2,4,6,7and9were shown to effectively induce osteogenic differentiation ofMSCs in vivo and in vitro. BMP-9is a important member of BMPs, inwhich has far stronger function to induce osteogenic differentiation thanthat of BMP2and BMP7. So it is possible to be applied in clinicaltreatment as a stronger osteoinductive factor in the near future.Bone regeneration and bone formation need the Coordinate regulationof many growth factors in the complex biological network. At present, although we do not quit know the osteoinductive mechanism of BMP-9inthe process of bone regeneration and bone formation. Based on BMP-9strong bone induction ability, we are urgent to hope it can play its role inclinical treatment as soon as possible. BMP-9has been confirmed itsimportant role in induce osteogenic differentiation and induce osteogenicdifferentiation in vivo using recombinant adenovirus as vector to mediateBMP-9. Recombinant adenovirus is a defective virus and can nottheoretically change the gene of host cells, so it is an ideal experimentalexpression system, however, the application of recombinant adenovirus inclinical has long time to study for its safety. Because of the lowimmunogenicity of BMPs and its just slight immunologic stimulation,BMPs can not provoke immunologic rejection. So the extraction andpurification of bone morphogenetic protein9applied to clinical can yet beregarded as a kind of simple and rapid method.The expression system from mammalian cells is an excellenteukaryotic expression system, and more than50%of the complex drugproteins were made from this system. Chinese hamster ovary-dihydrofolate reductase-deficient (CHO-dhfr-) is currently the preferredexpression system of restructuring glycosyl proteins production, and it isthe best eukaryotic genes express foreign host. It is easy for the synthesis ofexogenous protein in CHO and secreting to medium. The folding ofexpression proteins and the formation of disulfide bond are the same as the natural protein. BMP-9would be glycosylation after translation andsecreted into cells in the form of dimmer.The objective of this study is as following.(1) Reconstructing eukaryotic expression vector (pcDNA4/HisMax-BMP-9) by polymerase chain reaction;(2) constructing a stableeukaryotic expression system with CHO-dhfr-cells line and identifyingrhBMP-9after purification;(3) detecting the osteoinductive activity ofrhBMP-9by mesenchyme stem cells (C3H10) under the stimulated culture.The first, using the plasmid (padtrack-cmv-bmp9) as a template toamplify the total sequence of hBMP-9by polymerase chain reaction toobtain hBMP-9cDNA, and insert cDNA into plasmid (pcDNA4/His MaxA), finally obtain a new plasmid (pcDNA4/His Max-hBMP-9) foreukaryotic expression after identification including PCR, enzyme cutting,sequencing. The result show the hBMP-9gene would not be mutation andthe precision of reading frame, so we might undertake the next step.The second, Cotransfect tow plasmids (pcDNA4/His Max-hBMP-9and pSV2-dhfr) into dihydrofolate reductase (dhfr)-deficient CHO cells byliposomes, which two plasmids is ratio of six than one. The subsequentscreening by Zeocin (the concentration is100μg/ml) and the geneamplification in medium containing stepwise increments in methotrexatelevel such as0.02,0.08,0.32,1.0, and4.0μM. We finally obtain a stablemonoclonal cell strain expressing hBMP-9. Collect the nutrient medium supernatant from that of the monoclonal can express the highest rhBMP-9,subsequent purification by Ni-NTA His-Bind Resin columns, at last theidentification of protein by Western Blot. The highest expression level ofthat monoclonal is1.13μg/24h/106cells by ELISA; there are tow bands (themolecular weight is approximate32kD and50kD differently) showed byWestern Blotting and SDS-PAGE. The expression level of rhBMP-9islower than our expecting from the experimental data. It is possible that thesecretion of rhBMP-9is a dimer form in the process of secretion, and leadto difficulty in purification.The third part of the study is aimed at detecting osteogenic effect ofmesenchyme stem cells C3H10under the stimulated culture with rhBMP-9in vitro. Exponentially growing C3H10T1/2cells were seeded in24-wellplates with Dulbecco modified Eagle medium (DMEM) supplemented with10%FBS,100U of penicillin,100μg of streptomycin and rhBMP-9orrhBMP-2(100μg/ml) at37℃in5%CO2. The induction of alkalinephosphatase activity was assessed at seven days after cultivation; theinduction of mineralized matrix formation, osteocalcin and oesteopontinwere detected at twenty days. In addition, using C3H10(stimulated culture3d) with medical gelfoam obtained rhBMP-9(100μg/ml) to injectedBALB/c mice. Using the same concentration of rhBMP-2as experimentalcontrol. The results showed that, all osteogenic indexes of rhBMP-9wereless than rhBMP-2, and proteins couldn't form bone in vitro experiments. In summary, we can use bio-genetic engineering technology to build astable eukaryotic expression,and purify rhBMP-9to show biologicalactivity with specific protein tag. However, expression level of rhBMP-9and osteoinduction could not achieve the desired results, probably mainlydue to the lower protein expression level in screened monoclonal afterseveral passage, and a variety of existing forms of dimer (forming byrhBMP-9in CHO-dhfr-) lost part of activity rhBMP-9in purificationprocess. Although the experimental results was slightly regret, but the studyprovide a new way for clinical application of rhBMP-9as soon as possible.Therefore, whether obtain the more stable expression system by theinterference of the exogenous gene, and purify the active protein using thenew method, worthy of further reflection and research.
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