C端甲状旁腺相关肽的克隆及其对骨骺干细胞的调控作用

Cloning of C-terminal parathyroid hormone-related protein and its modulatory effects on precartilagenous stem cells

目的探讨C端甲状旁腺相关肽（PTHrp107-139）对体外培养的骨骺干细胞的调控作用以及与其他调控因子的关系。方法采用逆转录-聚合酶链反应（RT—PCR）方法克隆大鼠PTHrp全长中的107～139片段，并将其亚克隆至质粒pTRE2hyg上，构建新的质粒pTRE2hyg—PTHrp107-139，并将其与质粒pTet—on共转染分离纯化的骨骺干细胞，放入含有维生素c和地塞米松的全培养基中进行培养，分别加入0．1、0．5、2．0mg／L的强力霉素（Doxycycline）进行诱导，并设置空白对照。48～72h后抽提各组细胞的RNA进行逆转录，然后通过半定量PCR的方法检测增殖细胞核抗原（PCNA、SOX9等基因的表达变化，流式细胞仪检测细胞周期和凋亡。结果加入DOX组的PCNA、ColⅡ、血管内皮细胞生长因子（VEGF）、SOX9表达增加，骨骺干细胞数量明显增多，而Ptc、ColX、骨形态发生蛋白（BMP）-6表达减少。结论C端甲状旁腺相关肽可能有促进骨骺干细胞增殖并抑制其分化的作用，但对细胞凋亡无明显影响。

Objective To study the modulatory effects of parathyroid hormone-related protein （PTHrp） 107-139 on in vitro cultured precartilagenous stem cells and the relationship between PTHrp107-139 and other regulatory factors. Methods PTHrpl07_139 was cloned by RT-PCR, and then sub-cloned to plasmid pTRE2hyg in order to construct plasmid pTRE2hyg-PTHrp107-139 , which, together with pTet-on, was then co-transfected by lipofectamine 2000 to precartilagenous stem ceils. The stem cells were cultured in the medium supplemented with vitamin C and dexamethasone, and Tet-on was induced by different concentrations of doxycycline （0.1,0.5 or 2.0 μg/ml respectively）. Within 48 to 72 h,the RNA of each group was extracted by RT-PCR, then the changes of expression of genes such as Ihh, Ptc, PCNA, SOX-9 and BMP6 were examined by semiquantitative PCR. The cell cycle and apoptosis were examined by flow cytometry. Results The expression of PCNA, ColⅡ , VEGF and SOX-9 in doxycycline-treated groups was increased, and the number of precartilagenous stem cells increased significantly, and the expression of Ptc, Col X and BMP6 reduced. Conclusion C-terminal parathyroid hormone-related protein possibly promotes proliferation of precartilagenous stem cells and inhibits their differentiation, but has no effect on cell apoptosis.