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 osteoprotegerin gene

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是否有转录,同时通过Westernblot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39d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。

Objective In oder to treat periodontitis by using tissue engineering and gene engineering technology, the 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 By solation and culture of BMSC in vitro, the identified recombined plasmid was transiently transfected into BMSC by Lipofeetamine 2000 and OPG expression in BMSC was determined by RT-PCR and Western blot in 6 weeks. 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 GeneBank [gi：33878056]. OPG transcribing in BMSC was confirmed by RT-PCR and OPG sustainable expressing in BMSC was detected by Western blot in 39 days. Conclusion The transiently expression system of BMSC modified by OPG gene was successfully established.