骨形态蛋白9诱导干细胞骨向分化与环氧酶-2及PI3K/Akt的关系研究  

Study on relationship between BMP9-induced osteogenic differentiation and COX-2/PI3K/Akt in stem cells

作　　者：李洋[1,2] 陈前昭 邵英[1,2] 曾于桦 任文艳[1,2] 刘荣兴[1,2] 何百成[1,2] 

机构地区：[1]重庆医科大学药学院 [2]重庆市生物化学与分子药理学重点实验室,重庆400016

出　　处：《中国药理学通报》2017年第7期908-915,共8页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81372120)

摘　　要：目的研究骨形态蛋白9(BMP9)诱导干细胞成骨分化过程中对PI3K/Akt信号的激活与环氧酶-2(COX-2)的关系。方法利用组织化学染色法、化学发光法或定量PCR检测碱性磷酸酶(ALP)的水平,Western blot检测骨桥素(OPN)、骨钙素(OCN)、COX-2、Akt1/2和磷酸化Akt1/2(pAkt1/2)水平,RT-PCR检测COX-2的表达,用免疫组化或免疫荧光分别检测COX-2的表达水平以及Akt1/2的磷酸化水平,用茜素红染色检测钙盐沉积。结果 BMP9在C2C12细胞中明显增加ALP活性,促进OPN和OCN表达以及钙盐沉积;BMP9对Akt1/2总蛋白无明显影响,能明显增加Akt1/2磷酸化水平,PI3K抑制剂呈浓度依赖性减弱BMP9诱导ALP活性增加。BMP9在C2C12细胞中促进COX-2表达,抑制COX-2明显减弱BMP9在C2C12细胞中诱导ALP活性增加和钙盐沉积的作用。外源性过表达COX-2促进BMP9诱导p-Akt1/2水平增加,而抑制COX-2则明显减弱BMP9诱导Akt1/2磷酸化水平增加。结论 BMP9在诱导干细胞成骨化过程中对PI3K/Akt信号的激活可能与其促进COX-2表达有关。Aim To investigated the possible effect of COX-2 on the BMP9-induced activation of PI3K/Akt signal in progenitor cells.Methods The activity of alkaline phosphatase（ALP） was measured using histochemical staining or chemiluminescence.The m RNA level of ALP was determined using real-time PCR assay.The protein levels of osteopontin（OPN）,osteocalcin（OCN）,COX-2,Akt1/2 and phosphorylated Akt1/2 were detected by Western blot.The m RNA level of COX-2 was assayed with RT-PCR,and the mineralization was measured with Alizarin Red staining.Results The ALP activity was apparently increased by BMP9 in C2C12 cells,as well as the protein level of OPN and OCN.The mineralization was also markedly induced by BMP9 in C2C12 cells.BMP9 increased the level of phosphorylated Akt1/2 greatly,although no substantial effect was observed on total protein level of Akt1/2.The BMP9-induced ALPactivity was dramatically decreased by the inhibitor of PI3 K.The mR NA and protein level of COX-2 were both increased by BMP9 in C2C12 cells,and the BMP9-induced ALP activity and mineralization were greatly attenuated by the inhibitor of COX-2.The BMP9-induced phosphorylation level of Akt1/2 was increased by the exogenous expression of COX-2,but decreased by the inhibitor of COX-2.Conclusion Acti-vation of PI3K/Akt signaling may be a critical event in BMP9-induced osteogenic differentiation,and this process may be mediated by the BMP9-upregulated COX-2 in stem cells at least.

关 键 词：骨形态蛋白9 干细胞 成骨分化 成骨标志物 PI3K/AKT 环氧酶-2 

分 类 号：R341[医药卫生—基础医学] R329.2