| TGF-β s are multifunctional cytokines with a broad range of biological activities. TGF-β s belong to a family of related proteins, which demonstrate various degrees of amino acid sequence homology; this protein family is called the TGF-β superfamily. Other members of this family include the bone morphogenetic proteins (BMPs), inhibin, activin and Mullerian substance. In the past years, many studies have demonstrated there are close relations between TGF- P -like growth factors and bone repair. TGF- P s can make mesenchymal stem cells migrate and condense by chenotaxis, then stimulate them differentiate in a chondro- and osteoblast direction, which would correspondingly participate in bone formation process. Of such many TGF- P s, TGF- P 1, BMP2 and BMP? are paid more attentions for their abilities to induce bone formation.Recombitant human proteins and gene therapy are the two most important methods in application of TGF-β -like growth factors. In order to study the relation between bone formation and TGF-β -like growth factors, we cloned the genes of TGF-β1, BMP2 and BMP. TGF-β1 is expressed in E.coli and its active protein is produced; At the same time, pcDNA-BMP2 and pcDNA-BMP are cloned and recombinant adenoviruses Ad- TGF- P 1, Ad-BMP2 and Ad-BMP? are obtained; MSCs are separated and cultured in vitro, and applications of TGF- P 1, BMP2 and BMP? in gene therapy in vivo and ex vivo are studied. There are four parts in this research. 1. Cloning of TGF-β -like growth factor genesTGF-β 1 mature gene is obtained by PCR using pMAM- TGF-β 1 plasmid as a template; From osteosarcoma cell MG63 and human kidney cell 293,BMP2 and BMP? cDNA are cloned into T-vector respectively using RT-PCR. DNA sequence analysis showed that these cDNAs were almost identical with the published sequence. Cloning of these sequences lays the basis for the production of TGF- P 1 and gene therapy of the growth factors. 2.Expression of TGF- P 1 in E.coliGene engineering is one of the most important ways by which .proteins are obtained. TGF-β 1 mature gene was cloned into procaryotic expression vectors-pBV220 and pQE30 and monomeric forms of TGF- P 1 were expressed in E.coli DH5 a and Ml5 at high level. The dimeric protein, which is biologically active, was obtained by purification and refolding.S.Construction of eucaryotic expression vectors carrying TGF-β -like growth factor gene and expression in mesenchymal stem cells.BMP2 and BMP? genes were cloned into eucaryotic expression vector pcDNA3.1 and pcDNA-BMP2 and pcDNA-BMP? were constructed. At the same time, we isolated and cultured mesenchymal stem cells (MSCs) in vitro which was comfirmed by the cell surface markers. The plasmids pMAM- TGF-P 1 and pcDNA-BMP? were transfected into MSCs and the transfected cells were selected with G418, which can express TGF- P 1 and BMP? steadily. That laid the basis for gene therapy using these genes and provided the ideal seeds for tissue engineering.4. Preparation of recombinant adenovirus carrying TGF-β -like growth factor and study of ectopic bone formation.TGF-β 1, BMP2 and BMP? genes were cloned into the shuttle vector-pAdTrack; the bacterium BJ5183 was contransfected with the shuttle and the pAdEasy vector and the recombinant Ad plasmid was produced by homologous recombinantion. Recombinant adenovirus was packaged in HEK293 cells after the Ad plasmid was transfected. Ecotopic bone formation was studied using AdBMP2, the result demonstrated it could induce bone formation at ectopic site.In conclusion, engineering bacteria, which expressed TGF-β 1 at high level,were obtained and we set up the method of refolding and purification. BMP2 andBMP? genes were cloned and eucaryotic expression vector pcDNA-BMP2 and pcDNA-BMP7 were constructed. Ad TGF-β 1, AdBMP2 and AdBMP? were prepared. MSCs were successfully isolated and cultured. Gene therapy using TGF- P -like growth factor was primarily studied. These laid the substantial basis for using and studying of TGF- P -like growth factors.
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