CAJ | 학술논문

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.48～72 h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(PC-NA、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、Col X、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.
목적 탐토C단갑상방선상관태(PTHrp107-139)대체외배양적골후간세포적조공작용이급여기타조공인자적관계.방법 채용역전록.취합매련반응(RT-PCR)방법극륭대서PTHrp전장중적107～139편단,병장기아극륭지질립pTRE2hyg상,구건신적질립pTRE2hyg-PTHrp107-139병장기여질립pTet-on공전염분리순화적골후간세포,방입함유유생소C화지새미송적전배양기중진행배양,분별가입0.1、0.5、2.0mg/L적강력매소(Doxycycline)진행유도,병설치공백대조.48～72 h후추제각조세포적RNA진행역전록,연후통과반정량PCR적방법검측증식세포핵항원(PC-NA、SOX9등기인적표체변화,류식세포의검측세포주기화조망.결과 가입DOX조적PCNA、ColⅡ、혈관내피세포생장인자(VEGF)、Sox9표체증가,골후간세포수량명현증다,이Ptc、Col X、골형태발생단백(BMP)-6표체감소.결론 C단갑상방선상관태가능유촉진골후간세포증식병억제기분화적작용,단대세포조망무명현영향.
Objective To study the modulatory effects of parathyroid hormone-related protein ((PTHrp)107-139 on in vitro cultured precartilagenons stem ceils and the relationship between PTHrp107-139and other regulatory factors, nethods PTHrp107-139 was cloned by RT-PCR,and then sub-cloned to plas-mid pTRE2hyg in order to construct plasmid pTRE2hyg-PTHrp107-139, which, together with pTet-on, was then co-transfected by lipofectamine 2000 to precartilagenous stem cells.The stem cells were cultured in the medium supplemented with vitamin C and dexamethasone, and Tet-on was induced by different concen-trations 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 cytom-etry.Results The expression of PCNA,Col Ⅱ,VEGF and SOX-9 in doxycycline-treated groups was in-creased,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 precartilagenons stem cells and inhibits their differentiation, but has no effect on cell apop-tosis.