中文摘要：

目的：建立受强力霉素调控高表达外源基因的真核表达体系一永生化骨骺干细胞株，为调控甲状旁腺相关肽的不同片断在骨骺干细胞中的表达打下基础。方法：实验于2006-0912007-03在同济医学院矫形外科实验室完成。①实验材料：SD大鼠的乳鼠由华中科技大学同济医学院动物实验中心提供，雄鼠4只，雌鼠6只。②实验方法：取SD大鼠的乳鼠10只，显微镜下选取LaCroix环处的软骨及软骨前体细胞（骨骺干细胞），采用磁性细胞分选的方法，分离出原代骨骺干细胞，并用PCMVSV40质粒将其永生化。用脂质体介导法将pTet-on质粒转染已永生化的骨骺干细胞，通过G418筛选并经有限稀释法进行单克隆化，得到稳定转染的抗性克隆并将其分别扩增培养。将pTRE2hyg-luc质粒瞬时转染扩增后的单克隆细胞，加入终浓度为1mg／L的强力霉素诱导剂培养，48h后逐一检测每个细胞株的荧光素酶表达活性，逐一筛选强力霉素调控高表达外源基因的永生化骨骺干细胞株。③实验评估：将挑选出的第23号克隆扩增培养，加入O．01，O．1，O．5，1，5mg／L强力霉素进行诱导培养，并设置3组对照，一组为空白对照（蒸馏水），一组为未转染Tet-on系统的骨骺干细胞（均加入1mg／LO．1mg／强力霉素诱导），另一组为转染后的骨骺干细胞不加入强力霉素诱导。观察强力霉素对骨骺干细胞的最优诱导质量浓度。结果：第23号克隆的细胞经诱导后荧光素酶的表达活性为85790，而非诱导状态下该细胞株的活性为437，诱导后的活性增加196．3倍。强力霉素质量浓度梯度实验提示，强力霉素的最优诱导质量浓度是1mg/L左右，质量浓度过高可导致细胞死亡，过低时诱导效率降低。结论：永生化骨骺干细胞株可用于真核调控高表达，为研究甲状旁腺相关肽的不同功能片断与骨骺干细胞的分化机制的关系

英文摘要：

To establish a doxycycline-controlled eukaryotic expression system of highly expressed alien genes, Le. immortalized epiphysis stem cells, and a base for controlling the expression of various fragments of parathyroid hormone-related peptide （PTHrp）. METHODS： The experiment was carded out at the laboratory of the Department of Orthopaedic Surgery of Tongji Medical College from September 2006 to March 2007.①The neonatal SD rats, 4 male and 6 female, were selected at random from Animal Experimental Center of Tongji Medical University of Huazhong University of Science and Technology.②The chondrocytes and precartilagenous stem cells （epiphysis stem cells） of La Croix were collected from ten neonatal SD rats under the microscope. Primary epiphysis stem cells were harvested by immunomagnetic beads method and then immortalized with plasmid PCMVSV40. Plasmid pTet-on was transfected into immortalized epiphysis stem cells with lipofectamine 2000, and then the stably transfected positive clones were screened with G418 and subcultured by limiting dilution assay. The doxycycline at a concentration of 1 mg/L in the medium was added into monoclonal cells those were transiently transfected with plasmid pTRE2hyg-luc. Within 48 hours, the expression of luciferase of every cell line was detected individually, so that the doxycycline-controlled immortalized epiphysis stem cells of highly expressed alien genes was screened one by one.③The extracted No.23 cloned cells were sub-cultured and doxycycline at 0.01, 0.1, 0.5, 1, 5 mg/L concentrations was added to culture, and then three control groups were set as bland control （distilled water）, non-transfected Tet-on epiphysis stem cells （1 mg/L doxycycline）, and transfected epiphysis stem cells. The optimized concentration of epiphysis stem cells induced by doxycycline was observed. RESULTS： The activity after the induction increased by 196.3 times, for the expression of luciferase of the induced No. 23 cloned cells was 85790, while the activity of the non-induced