【作者】 郭国宁；

【导师】 周跃；

【作者基本信息】 第三军医大学 ， 外科学， 2005， 博士

【摘要】 骨不连、骨缺损、脊柱融合的治疗实际上是促进骨修复与重建的过程。骨诱导因子与细胞膜相应受体结合,通过信号转导,导致成骨。实验和临床研究均证实,骨不连的主要原因是成骨障碍,伴有骨折断端大量纤维瘢痕组织形成、并充填骨折断端,瘢痕组织主要由成纤维细胞构成,成纤维细胞和成骨细胞都起源于间充质干细胞,如果改变成纤维细胞的特征,使之具有特征性的成骨表型,具有非常重要的理论和临床意义。目前发现成骨细胞仅比成纤维细胞多表达两个基因,即骨钙素和Cbfa1。因此,从遗传学上讲,成骨细胞可以认为是更高级的成纤维细胞(sophisticated fibroblast)。Cbfa1 是成骨细胞的特异性转录因子。Cbfa1 调节成骨分化标志基因的表达,促进骨分化和骨形成。在骨诱导因子与Cbfa1 的关系中,一方面,骨诱导因子通过Cbfa1促进成骨;另一方面,Cbfa1 也可上调骨诱导因子或其受体的表达正反馈调节成骨,可以说Cbfa1 是多种骨诱导因子诱导成骨的共同信号分子,如果能将Cbfa1 基因转移间充质源性细胞就可能成骨。已经有研究证实:NIH3T3 细胞在逆转录病毒/Cbfa1 感染后,出现了VEGF 的基因和蛋白的表达,在BMP 的作用下向成骨表型转变。正是鉴于成纤维细胞和成骨细胞相同的起源、很小的基因表达差异、Cbfa1对于成骨细胞的重要性及特殊性引入本研究。研究目的: 体外Cbfa1基因转移能否改变成纤维细胞的表型而向成骨表型转变;体内Cbfa1对骨缺损和脊柱融合有无治疗作用。主要的方法及技术路线: 1. 脂质体法介导Cbfa1真核表达质粒转染NIH3T3,RT-PCR、生化方法、免疫细胞化学染色、Western-blot检测成骨标志基因Cbfa1、OCN、Type Collagen I、ALP、Bsp在转录水平及蛋白水平的表达。2. 制作小鼠的颅骨缺损模型,实验分为4组:(1) Cbfa1工程成纤维细胞移植组;(2)空白对照组;(3) 明胶海绵移植组;(4) NIH3T3移植组。术后8w大体、X线观察骨修复的情况。3. 构建AdEasy1/Cbfa1 病毒载体。PCR 扩增Cbfa1 的全长cDNA,T-A 克隆,亚更多还原

【Abstract】 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.更多还原