过表达Runx2促进C2C12细胞成骨分化

Overexpression of Runx2 Induces Osteogenic Differentiation in C2C12 Cells

利用Tet-on(Tetracycline-on)基因表达系统,通过强力霉素(doxycycline,DOX)诱导Runx2基因在C2C12细胞中的表达,探究Runx2促成骨分化功能,为其分子机制的研究提供一个理想的实验平台.先后将调控质粒pTet-on和反应质粒pTRE-Flag-Runx2转染入C2C12细胞,并用G418和潮霉素分别进行2轮筛选,运用实时荧光定量PCR选择对强力霉素诱导敏感的细胞克隆.用不同浓度DOX诱导C2C12/Tet/pTRE-Flag-Runx2细胞,蛋白免疫印迹检测Runx2的表达,确定DOX的最佳诱导浓度与时间,并检测C2C12细胞的成骨分化能力.结果表明,诱导细胞最佳DOX浓度为10μg/ml;最佳诱导时间为12h;诱导后Runx2基因高表达,C2C12细胞向成骨方向分化(P<0.05).成功建立Tet调控Runx2基因表达C2C12细胞系,为进一步研究Runx2基因功能分子机制提供理想的细胞模型.

A tet-on regulating system of the functional Runx2 expression was established in mouse myoblast cell line C2C12.A pTet-on regulating plasmid was first transfected into C2C12 and selected with G418 for stable clones.Then the pTRE-Flag-Runx2 was transfected into the obtained positive C2C12/Tet cells and selected with hygromycin.When Dox was used to induce the expression of Runx2,a sensitive clone was screened through quantitative RT-PCR.The optimal Dox induction condition of 10 μg/ml for 12 hours was determined by quantitative RT-PCR and Western blot assays.ALP staining and Alizarin red（AZR） staining after Dox treatments were used to evaluate the capability of osteogenic differentiation.The levels of differentiation of C2C12/Tet/pTRE-Flag-Runx2 cells were significantly higher than those of native C2C12 cells or C2C12/Tet/pTRE cells（P0.05 or 0.01）.Our Tet-on regulated Runx2 expression system in C2C12 cells may provide a useful tool for the further study of Runx2 functions.