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论文摘要

骨保护素修饰的Beagle犬骨髓基质细胞表达体系的建立与鉴定

Establishment and identification of transiently expression system of bone marrow stromal cells modified by steoprotegerin gene

作者:赵春晖 操小马 梅陵宣

Author:ZHAO Chun-hui1, CAO Xiao-ma1,2, MEI Ling-xuan1

收稿日期:2008-12-25          年卷(期)页码:2008,26(06):673-

期刊名称:华西口腔医学杂志

Journal Name:West China Journal of Stomatology

关键字:成骨细胞,骨髓基质细胞,基因表达,

Key words:osteoblasts,bonemarrowstromalcells,geneexpression,

基金项目:

国家自然科学基金资助项目(C03031101);安徽省教育厅自然科学基金资助项目(2004kj219);安徽医科大学校基金资助项
目(2003zr01)

中文摘要

目的利用pSecTag2/B-OPG真核分泌表达穿梭载体,建立经骨保护素基因(OPG)修饰的Beagle犬骨髓基质细胞(BMSC)瞬时表达体系并检测其表达能力,为基因工程与组织工程联合治疗牙周病提供细胞基础。方法体外分离、培养Beagle犬BMSC,通过脂质体法将已鉴定的目的基因瞬时转染至BMSC,并用RT-PCR鉴定OPG是否有录,同时通过Western blot方法检测OPG在6周内的表达水平。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GeneBank中[gi:33878056]提供的序列一致;鉴定正确的重组质粒在BMSC中有转录,并且在39 d内都明显有OPG表达。结论建立了骨保护素基因修饰的骨髓基质细胞瞬时表达体系。

英文摘要

Objective In oder to treat periodontitis by using tissue engineering and gene engineering technology, he article established an transient expression system of bone marrow stromal cells(BMSC) modified by osteoprotegerin OPG) gene and detected its expression using eukaryotic secreted expression pSecTag2/B-OPG plasmid. Methods y solation and culture of BMSC in vitro, the identified recombined plasmid was transiently transfected into BMSC y Lipofectamine 2000 and OPG expression in BMSC was determined by RT-PCR and Western blot in 6 weeks. esults The fragments of the recombinant plasmid digested with Hind Ⅲ,EcoR I and BamHⅠ and examined by 0 g/L agarose electrophoresis, were consistent with predicted size. The sequence of OPG was identical to the equence provided by GeneBank [gi:33878056]. OPG transcribing in BMSC was confirmed by RT -PCR and OPG ustainable expressing in BMSC was detected by Western blot in 39 days. Conclusion The transiently expression ystem of BMSC modified by OPG gene was successfully established.

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