SDF-1/CXCR4在BMP9介导C2C12细胞成骨分化过程中的作用研究

The Role for SDF-1/CXCR4 Signal Axis in BMP9-induced Osteogenic Differentiation of C2C12 Cells

为了观察SDF-1/CXCR4信号轴在BMP9促C2C12细胞成骨分化过程中的作用,通过重组腺病毒过表达BMP9,检测对C2C12细胞中SDF-1及受体CXCR4 mRNA和蛋白表达水平的影响;同时利用重组腺病毒或中和抗体干扰SDF-1/CXCR4,与BMP9先后作用于C2C12细胞,通过定量测定碱性磷酸酶(ALP)、染色测定ALP表达、免疫细胞化学测定骨钙蛋白(OCN)表达、茜素红S染色测定钙盐沉积、Real-time PCR检测成骨相关转录因子Runx2和Osx的表达、Western blot检测成骨分化信号通路MAPK和Smad的变化。结果显示,BMP9能明显抑制C2C12细胞中SDF-1、CXCR4的表达(P<0.01),且具有剂量和时间依赖性;预先干扰SDF-1/CXCR4能明显影响由BMP9介导的早、中期成骨标志物ALP、OCN及早期转录因子Runx2、Osx的表达(P<0.01)和MAPK、Smad信号通路相关蛋白的变化(P<0.05);外源性SDF-1并不能影响晚期成骨标志物钙盐沉积。提示SDF-1/CXCR4信号轴在由BMP9介导的C2C12细胞成骨分化早、中期过程中发挥重要作用。

To elucidate the role of SDF-1/CXCR4 signal axis during BMP9-incuced osteogenic differen- tiation in C2C12 cells, BMP9 was introduced by using recombinant adenoviruses assay. The mRNA and protein expression levels of SDF-1 and CXCR4 induced by Ad-BMP9 were detected in C2C12 cells. At the same time, the recombinant adenovirus and neutralizing antibody were used to perturbing the SDF-1/CXCR4 signal axis in C2C 12 cells before or after the addition of BMP9. The alkaline phosphatase （ALP） quantitative assay and fast blue RR salt staining were used to determine the expression of ALP. Immunocytochemistry was used to determine the expression of osteocalcin （OCN）, while calcium deposition was determined by alizarin red S staining. The expression of the osteogenic transcription factor Runx2 and Osx were detected by real time PCR. Western blot was used to detect the change of osteogenic differentiation signaling pathway MAPK and Smad. The results showed that BMP9significantly inhibited SDF-1 and CXCR4 expression （P〈0.01） in C2C12 cells, in a dose- and time-dependent. Pretreatment of C2C12 cells with SDF-1/CXCR4 could significantly affect the early and mid osteogenic markers ALP, OCN, the transcription factors of Runx2, Osx expression （P〈0.01）, and the Smad, MAPK signaling pathway （P〈0.05）. Addition of exogenous SDF-1 did not affect the changes of the late osteogenic marker calcium deposi- tion. Our data indicated a co-requirement of the SDF-1/CXCR4 signal axis in BMP9-induced the early and mid- process of osteogenic differentiation of C2C 12 cells.