p38蛋白激酶参与BMP9诱导的C3H10T1/2细胞成骨分化

p38 Kinase Participated in BMP9-induced Osteogenic Differentiation of C3H10T1/2 Mesenchynal Stem Cells

目的:初步分析丝裂原活化蛋白激酶p38在BMP9诱导间充质干细胞C3H10T1/2成骨分化过程中的作用。方法:利用BMP9重组腺病毒感染C3H10T1/2细胞,Western blot检测p38激酶总蛋白表达水平和磷酸化水平。p38的特异性抑制剂SB203580抑制p38活性或RNA干扰抑制p38表达后,分析ALP活性变化,利用茜素红S染色检测钙盐沉积,Real Time PCR检测Smad6和Smad7的mRNA表达水平,动物实验确认在RNA干扰p38蛋白激酶后,对于BMP9诱导的C3H10T1/2细胞异位成骨的影响。结果:BMP9不影响p38激酶的蛋白表达水平,但却可以促进p38激酶的磷酸化;p38抑制剂SB203580可剂量依赖性地抑制由BMP9诱导的C3H10T1/2细胞的碱性磷酸酶(alkaline phosphatase,ALP)活性,RNA干扰导致p38基因沉默同样也可抑制BMP9诱导的ALP活性,但抑制效应不及SB203580;SB203580还能够抑制BMP9诱导的C3H10T1/2细胞的钙盐沉积;BMP9的靶基因Smad6和Smad7的mRNA表达也能被SB203580所抑制;干扰p38蛋白激酶可抑制BMP9诱导的C3H10T1/2细胞在裸鼠皮下异位成骨。结论:p38蛋白激酶参与了BMP9诱导的C3H10T1/2成骨分化。

Objective：Analysis the functional role of p38 kinase in BMP-9-induced osteogenic differentiation of C3H10T1/2 mesenchymal stem cells.Methods：C3H10T1/2 cells were infected by recombinant adenovirus expressing BMP9,then the total protein level and phosphorylated form of p38 kinase were determined by Western blot.After treatment C3H10T1/2 cells with p38 specific inhibitor SB203580 or using RNA interference to silence expression of p38,the early osteogenic marker ALP activity was detected by quantitative and staining assay,later osteogenic marker calcium deposition was determined by Alizarin Red S staining,expression level of Smad6 and Smad7 was analyzed by Real time PCR.Animal assay was carried out to confirm that whether p38 silence can result in inhibition of entopic bone formation induced by BMP9 in vivo.Results： BMP9 did not change total protein level of p38,however,BMP9 increased the phosphorylated form of p38 kinase.P38 specific inhibitor SB203580 dose-dependently decreased ALP activity induced by BMP9 of C3H10T1/2 cells.Gene silence of p38 by RNA interference also led to a reduction of ALP activity.Furthermore,SB203580 markedly inhibited calcium deposition,reduced BMP9-induced expressions of Smad6 and Smad7.Moreover,p38 gene silence was also showed to inhibit entopic bone formation induced by BMP9 in vivo.Conclusion： The p38 kianse may invovlve in BMP9 induced osteoblast commitment of C3H10T1/2 mesenchymal stem cells.