pTet-on骨骺干细胞株的建立和pTRE-甲状旁腺相关蛋白（1—36）反应质粒的构建  

Establishment of pTet-on rat precartilaginous stem cell strains and construction of pTRE-PTHrP （1-36） responsive plasmids

作　　者：张树威[1] 陈安民[1] 李明辉[1] 祁军[1] 宋登新[1] 祝文涛[1] 廖晖[1] 郭风劲[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院骨科,武汉430030

出　　处：《中华创伤杂志》2008年第7期564-568,共5页Chinese Journal of Trauma

基　　金：国家自然科学基金资助项目（30571872）;教育部博士点基金资助项目[教技发中心函（2005）216]

摘　　要：目的建立pTet—on骨骺干细胞株，克隆甲状旁腺相关蛋白[PTHrP（1—36）]基因并构建反应质粒pTRE—PTHrP（1—36）。方法用脂质体介导的基因转染技术将pTet—on质粒转染于永生化骨骺干细胞，G418筛选得到稳定克隆，再瞬时转染pTRE-2Hyg—Luc质粒并筛选出高水平诱导、低背景表达的永生化骨骺干细胞系。以RT—PCR方法获得带有酶切位点PTHrP（1—36）基因片段，与载体pTRE-2Hyg均双酶切后连接，转化扩增后对重组质粒进行提取和酶切、测序鉴定。结果筛选到1株诱导后荧光素酶的表达活性增加50倍的骨骺干细胞系；经酶切图谱分析和DNA序列测定证实目的基因已经插入重组质粒。结论筛选得到的永生化骨骺干细胞株受强力霉素诱导后能够低背景、高水平表达；成功克隆PTHrP（1—36）基因并构建了反应质粒pTRE—PTHrP（1—36），为进一步精确调控PTHrP（1—36）基因的表达奠定了基础。Objective To establish a doxycycline-controlled immortalized pre-cartilaginous stem cells （IPCSCs） strains, clone parathroid hormone-related peptide（ PTHrP（1-36） ] gene and construct responsive plasmid, pTRE-PTHrP （1-36）. Methods Plasmid pTet-on was transfected into IPCSCs by using LipoinfectaminTM 2000 and then the stable clones were obtained by G418 screening. The doxycycline was added into the medium of monoclonal cells that were transiently transfected with plasmid pTRE-2Hyg-Luc. The total RNA was extracted from PCSCs and the PTHrP（1-36） gene obtained by RT-PCR method. Then, the PTHrP （1-36） gene was subcloned to plasmids of Tet-responsive element with the selection marker of hygrornycin pTRE-2Hyg to construct recombinant eukaryotic expression plasmid pTRE- PTHrP（1-36）. After transferred into E. coli-DH5α, the clone was amplified, the recombinant plasmids were purified and identified by double-enzyme digestion. Results The doxycycline induced IPCSCs line was obtained, with 50 times higher than the non-induced cell line. Double enzyme digestion analysis and sequencing showed that the target gene was cloned into recombinant plasrnid. Conclusions The induced IPCSCs line can be used to highly express alien genes. The responsive plasmid containing PTHrP （1-36） gene may be promising for rigorous control of PTHrp （1-36） gene expression.

关 键 词：骨骺 强力霉素 基因表达调控 甲状旁腺相关蛋白 

分 类 号：R68[医药卫生—骨科学]