The Study Of Modulation Of The LMP-3 Gene Transfer On Marrow Stromal Cells And It's Role In Treating Bone Defect

Objective The bone defect caused by trauma and bone disease is one of difficult problems of clinical work. Following the progress of DNA recombiant and transfer technology and tissure engineer technology, the treatment of bone defect obtained rapid development. LMP as a bone formation inducted protein was discovered recently, it's proved that LMP-1 and 3 had the role of osteogenesis, it's a positve regulator factor in osteoblast differentiation program, it can induce the secretion of multiple osteoinductive factor and induce the osteocalcin secretion and bone nodular formation. LMP play an important role in the progress of bone mineralization. In this study, the eukaryotic expression plasmid of LMP-3 was constructed, and it's effect on bone marrow stromal cells was stuied. At the same time, the plasmid combinated gelatin sponge was implante to the defect of rabbit radial bone and to investigated it's osteogenesis in vivo.Methods Applying the technology of PCR to amplify the functional regeion of LMP-3, and subclonging it to the the eukaryotic expression plasmid of pEGFP-N1, the recombianted expression plasmid was constructed and identified by restriction enzyme and sequencing. The plasmid of pEGFP-N1- LMP-3 was transferred to bone marrow stomal cells by liposome, the phenotype changes of BMSC was observed ,the expression of osteogenesis marker gene include OCN,Type Collagen I,ALP and Bsp was detected by RT-PCR, Biochemistry detect ALP activity and immunity detect OCN. The formation of mineralization nodule was observed by alizarin red stain. The expression of Type Collagen I was observed by immunocytochemical stain. The animal model of rabbit radial defect was made, after plasmid combinated gelatin sponge was implanted tothe defect of rabbit radial bone, the bone healing was evalulated by X-ray and HE stain.Results The gene fragment encoding LMP-3 was amplified correctly by PCR. Enzyme digestion and identical sequencing showed that the recombinant eukaryotic expression plasmid was right constructed. Rabbit's bone marrow stomal cells was cultured successfully, after transfected by pEGFP-N1- LMP-3, the expression of OCN,Type Collagen I,ALP,Bsp was detected by RT-PCR.The formation of mineralization nodule was observed by alizarin red stain, the ALP and OCN was also found more expression. The expression of Type Collagen I was more obviously than control group. The animal model was constructed successfully, the bone restore was proved by X-ray and histological observation in experimental group.Conclusions Recombinant eukaryotic expression plasmid of LMP-3 has been successfully obtained, suggesting a foundation for the further studies on the bone formation. After transfected pEGFP-N1- LMP-3 , BMSC could secret osteogenesis marker gene , the functional region of LMP-3 gene could induce the phenotype change of BMSC , the functional region had the same osteogenesis role as it's total cDNA. By the scaffold of gelatin sponge and seed cells of BMSC, the gene LMP-3 could promot the healing of bone defect effectivel, it may be a new treat method of bone defect.