| Therapy of bone nonunion, bone defect and spinal fusion is the process of promoting bone repair which is the results of signal transduction of osteoinductive factor binding to corresponding receptor. Because both fibroblast and osteoblast derived from mesenchymal stem cell and have the same gene apart from Cbfa1 and OCN, the osteoblast is the more sophisticated fibroblast. The scar is mainly made of fibroblast which prevent bone repair. If the phenotype of fibroblast can be changed to osteoblast, it will have important significance in theory and clinic. Core binding factor a is the osteoblastic specific transcriptional factor which regulate osteogenesis marker gene differentiation and promote bone formation. Relationship between Cbfa1 and osteoinductive factor is: osteogenesis effect of osteoinductive factor is through Cbfa1, on the other hand, Cbfa1 can upregulate osteoinductive factor and their receptor to promote bone formation. It can be said Cbfa1 is the common signal of inductive bone formation by various osteoinductive factors. There maybe bone formation if Cbfa1 gene transfer to mesenchymal cells. It has been confirmed that there appear VEGF expression after Cbfa1 retrovirus infect NIH3T3 and osteogenesis phenotype after BMP treat fibroblast. Just respecting the common origin, little gene difference of fibroblast and osteoblast and the importance of Cbfa1 for osteoblast, we do this study. Objective Whether Cbfa1 gene transfer can change the phenotype of fibroblast to osteoblast in vitro and have positive effects on bone defect and spinal fusion in vivo. Main methods and techniques: 1. After Cbfa1 plasmid transfect NIH3T3 by liposome, RT-PCR detect the expression of osteogenesis marker gene of Cbfa1,OCN,Type Collagen I,ALP,Bsp; Biochemistry detect ALP activity; Immunocytochemical stain detect expression of OCN; Western-blot screen the protein expression of Cbfa1. 2. To make skull defect model of mouse which is divided into 4 groups: (1) Cbfa1 engineering fibroblast transplant; (2) blank control; (3) gel foam transplant; (4) NIH3T3 transplant. Evaluate the repair effects of skull defect by specimen observation and X-Ray results at 8 week. 3. To construct AdEasy1/Cbfa1 recombined virus vector. PCR amplify the full cDNA of Cbfa1, T-A clone, then subclone to the shuttle plasmid of pAdTrack, homologous recombination with pAdEasy1 in BJ5183 bacteria, packet in 293T cells to produce virus. Construct the control virus of AdTrack/GFP in the same way. Purify the virus by CsCl gradient centrifuge. Verify the recombined virus by PCR and Western-blot. Determine the tilter and infection efficiency of the recombined virus by GFP tracer. 4. To culture rabbit skin fibroblast by trypsinization, then verify by immuno-cytochemical stain. After AdEasy1/Cbfa1 infect the cell, RT-PCR detect the the expression of osteogenesis marker gene of Cbfa1, OCN, Type Collagen I, ALP, Bsp; Biochemistry detect ALP activity; Western-blot screen the protein expression of Cbfa1; Noeud stain ALP and mineral; Radioimmunity detect OCN. 5. To make rabbit intertransverse fusion model which is divided into 5 groups: (1) Blank control; (2) AdEasy1/GFP control; (3) AdEasy1/Cbfa1 injection; (4) AdEasy1/Cbfa1 infection fibroblast transplant; (5) GFP tracer. Evaluate the repair effects of intertransverse fusion by specimen observation, X-Ray results and HE stain at 4 and 8 week. Main results and conclusions: 1. After Cbfa1 transfect NIH3T3, the gene and protein expression of osteogenesis marker gene were detected. It indicates Cbfa1 gene transfer can change the phenotype of NIH3T3 to osteoblast. 2. There is new bone formation of the test nevertheless no of control. It indicates that the osteogenesis phenotype of NIH3T3 have the repair effect on bone defect in vitro. 3. Successfully construct AdEasy1/Cbfa1, its infection tilter is 2.1×1013Pfu/ml and has high infection efficiency, which can be used in the study of gene therapy. 4.Successfully cultured rabbit skin fibroblast which is verified positive by immunocytochemical stain. After AdEasy1/Cbfa1 infect cell, there were osteogenesis marker gene expression and protein expression. It indicates that AdEasy1/Cba1 switch on the transcription of osteogenesis gene and change the phenotype of fibroblast toosteogenesis. 5. There were intertransverse fusion of the test animal model nevertheless no of control. It indicates using AdEasy1/Cbfa1 to treat spinal fusion is feasible which provide easy and new method for spinal fusion. and the method of region injection have the merit of easy process and .microtrauma, which can represent the idealest microtrauma spinal fusion.
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