摘要
目的研究环氧酶-2(COX-2)在骨形态蛋白9(BMP9)诱导间充质干细胞(MSCs)成骨分化过程中的作用,以及COX-2影响BMP9功能的可能机制。方法采用定量PCR、蛋白印迹和免疫细胞化学染色分析BMP9对COX-2表达的影响。采用化学发光法检测碱性磷酸酶(ALP)的活性,用RT-PCR法检测Smad6、Smad7 mRNA表达水平,用蛋白印迹检测Runx2、Dlx-5、Smad1/5/8及磷酸化Smad1/5/8蛋白水平。通过体内异位成骨实验检测COX-2对BMP9诱导MSCs成骨分化的影响。利用萤光素酶报告质粒检测BMPs/Smads信号活化程度。结果 BMP9明显诱导COX-2表达,抑制COX-2酶活性或沉默COX-2均抑制BMP9诱导C3H10T1/2细胞ALP活性增加。沉默COX-2明显抑制BMP9诱导C3H10T1/2细胞表达Runx2和Dlx-5,以及BMP9诱导的C3H10T1/2细胞异位成骨。沉默COX-2抑制BMPR-Smad报告质粒萤光素酶活性,降低Smad1/5/8的磷酸化水平,以及抑制Smad6和Smad7的mRNA表达。结论 COX-2对BMP9诱导MSCs成骨分化具有重要调节作用,其机制可能与COX-2调节BMPs/Smads信号转导有关。
Aim To investigate the role of COX-2 in BMP9 induced osteogenic differentiation,and the pos-sible mechanism underlying this function of COX-2. Methods We introduced real-time PCR, Western blot, and immunocytochemical staing to detect the effect of BMP9 on COX-2 expression.We employed chemiluminescence technique to assay ALP activities, RT-PCR to detect the expression of Smad6 and Smad7 , and Western blot to measure the expression of Runx2, Dlx-5,total Smad1/5/8,and phosphorylated Smad1/5/8.Finally,BMPR-Smad luciferase reporter assay was applied to measure the activation of BMPs/Smads signaling.Results BMP9 could induce the expression of COX-2 in C3H10T1/2 cells.Either inhibiting enzy-matic activity of COX-2 or knockdown of the expression of COX-2 reduced the BMP9 induced ALP activities in C3H10T1/2 cells,and COX-2 knockdown also inhibited the ectopic bone formation induced by BMP9 in C3H10T1/2 cells.Moreover,COX-2 knockdown inhibi-ted BMPR-Smad reporter activities and the phosphoryl-ation of Smad1/5/8,so did the expression of Smad6 and Smad7 .Conclusion COX-2 may play an impor-tant role in BMP9 induced osteogenic differentiation in MSCs by regulating the BMPs/Smads signaling trans-duction.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2014年第7期1006-1011,共6页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81071462
81372120)
