重组质粒pSecTag2/B-OPG在Beagle犬骨髓基质细胞中表达与鉴定  

Expression and identification of recombinant plasmid of pSecTag2/B-OPG in Beagle dog bone marrow stromal cells in vitro

作　　者：操小马[1] 赵春晖[1] 梅陵宣[1] 

机构地区：[1]安徽医科大学口腔医学院,合肥230032

出　　处：《安徽医科大学学报》2007年第2期140-143,共4页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金项目(编号:C03031101);安徽省教育厅自然科学基金项目(编号:2004kj219);安徽医科大学校科研基金项目(编号:2003zr01)

摘　　要：目的转染骨保护素（OPG）基因至Beagle犬骨髓基质细胞（BMSCs）中，并检测其表达能力，为基因工程技术治疗牙周病提供物质基础。方法穿刺抽取成年Beagle犬骨髓2～3ml，通过全骨髓贴壁法获取BMSCs。传代至第3代，脂质体瞬时转染经鉴定的重组pSecTag2／B-OPG，并用RTPCR、Western blot检测OPG的表达水平。结果重组质粒pSecTag2／B-OPG经HindⅢ单酶切及EcoRⅠ、BamHⅠ双酶切，电泳显示切下的片段均与预期大小相符，经测序证实此基因与GenBank中提供的序列[gi：33873056]一致；RTPCR、Western blot结果显示：重组pSecTag2／B-OPG在骨髓基质细胞中有表达。结论成功建立了OPG基因修饰的骨髓基质细胞瞬时基因表达体系。Objective To identify expression of human osteoprotegerin（OPG） in Beagle dog bone marrow stromal ceils（BMSCs）. Methods Adult Beagle dog bone marrow about 2 - 3 ml were drawn by puncture. Cells were isolated by a whole bone marrow adherence. After expanding and culture 3 passages, the identified recombined plasmid was transiently transfected into BMSCs by Lipofectamine 2000 and OPG expression in BMSCs was determined by RT-PCR and Western blot. Results The fragments of the recombinant plasmid digested with Hind Ⅲ, EcoR Ⅰ and BamH Ⅰ and examined by 10 g/L agarose electrophoresis were consistent with predicted size. The sequence of OPG was identical to the sequence provided by GenBank[ gi ：33878056]. OPG expressing BMSCs was confirmed by RTPCR and Western blot. Conclusion The transiently expression system of BMSCs modified by OPG gene is successfully established.

关 键 词：成骨细胞 骨髓细胞 克隆 分子 基因表达 牙周疾病/治疗 骨保护素 BEAGLE犬 

分 类 号：R394.3[医药卫生—医学遗传学] R392.11[医药卫生—基础医学]