hBMP2和hVEGF165双基因修饰组织工程骨的构建及检测.DOC

hBMP2和hVEGF165双基因修饰组织工程骨的构建及检测 范伟，张弓 （重庆医科大学附属第一医院骨科，重庆 400016） 【摘要】 构建hBMP2和hVEGF165双基因修饰的组织工程骨并检测其基因表达情况。方法：构建pEGFP/hVEGF165和pEGFP/ hBMP2两种真核表达质粒，并用阳离子脂质体法转染至兔成骨细胞，再以脱蛋白骨（DPB）为支架，体外构建多基因修饰的组织工程骨，同时采用RT-PCR和Western Blot对组织工程骨中hVEGF165和hBMP2基因的表达情况进行检测。结果：转染hBMP2和hVEGF165的成骨细胞可以在DPB上贴附生长，并且两种基因的体外表达时间可持续3周左右。结论：成功构建hBMP2和hVEGF165双基因修饰组织工程骨，并且能有效表达hBMP2和hVEFGF165基因，为进一步观察其在体内的再血管化和成骨活性，用于大段骨缺损修复奠定了实验基础。 【关键词】组织工程骨，血管内皮细胞生长因子，骨形态发生蛋白，基因表达 Construction and detection of dual-gene-modified tissue engineering bone with the hBMP2 and hVEGF165 FAN Wei，ZHANG-Gong （Department of Orthopaedics,the First Affiliated Hospital, Chongqing Medical University Chongqing，400016） [Abstract] Objective: Construction of dual-gene-modified tissue engineering with hBMP2 and hVEGF165，and detection the gene expression of hVEGF165 and hBMP2. Method: This experiment is to construct two kind of eukaryotic expression plasmid, pEGFP/hBMP2 and pEGFP/VEGF165, which were both transfected into osteoblasts , and then build a dual-gene-modified tissue engineering bone in vitro with the Deproteinated Bone, DPB, as the frame. At he same time, we detected the gene expression of hVEGF165 and hBMP2 with RT-PCR and Western blot. Results: Osteoblasts transfected eukaryotic expression plasmids of pEGFP/hBMP2 and pEGFP/VEGF165 could be growthed in DPB, and both the hBMP2 and hVEGF165 could continue to express three weeks. Conclusion: The hVEGF165 and hBMP2 dual- gene- modified tissue engineering bone were constructed successfully, the genes of hVEGF165 and hBMP2 could be effective expressed in this tissue engineering bone, thus laying an experimental foundation for further observation of its revascularization and bone formation in vivo and for the repair of large bone defect. 【Key words】 Osteoblasts，Tissu Engineering?Bone，VEGF，BMP，Gene Expression 骨骼组织因创伤、肿瘤、退行性改变等造成的骨缺损在临床中十分常见，对缺损的修复主要采用骨移植的方法进行治疗。现有治疗手段均存在某些缺陷，组织工程学技术为骨缺损修复提供了新的途径。脱蛋白骨（deproteinated bone，DPB）具有天然网状孔隙系统，原骨盐支架的三维结构，其组成及结构符合生理要求，具有良好的组织相容性，有利于细