激活Shh信号通路促进BMP9诱导小鼠成肌细胞C2C12向成骨分化  

Activation of Shh Signaling Enhances BMP9-induced Osteogenic Differentiation in C2C12 Cells

作　　者：李丽[1] 欧欣颖 吉彩霞[1] 刘晓骅 何通川[1] 罗进勇[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016

出　　处：《中国生物化学与分子生物学报》2015年第5期481-488,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.81272006)~~

摘　　要：近年来骨组织工程技术迅猛发展,小鼠成肌细胞C2C12因其来源广泛等优点可望成为有效的种子细胞应用于组织工程.然而,对于C2C12细胞的成骨分化机制仍需深入研究.为了观察Sonic hedgehog(Shh)信号通路对骨形态发生蛋白9(bone morphogenetic proteins 9,BMP9)诱导的C2C12细胞成骨分化的影响,构建过表达腺病毒Ad-Shh,并作用于BMP9处理的C2C12细胞,检测碱性磷酸酶(alkaline phosphatase,ALP)的变化,茜素红S染色检测钙盐沉积,RT-PCR检测Shh、骨桥蛋白(osteopontin,OPN)、骨钙素(osteocalcin,OCN)、Runx2、Dlx5、Id1和Id2基因表达,Western印迹检测Shh、OPN、OCN、Runx2和Dlx5的蛋白质表达,Micro-CT和H&E染色检测裸鼠皮下异位成骨包块情况.结果表明,活化Shh信号通路可促进BMP9诱导的C2C12细胞早晚期成骨分化,以及裸鼠皮下异位成骨.体内外实验证明,Shh信号通路能促进BMP9诱导小鼠成肌细胞C2C12向成骨分化.Bone non-union is a serious disease in skeletal system. Recently,the rapid development of tissue engineering technology has been considered as an efficient method to improve the cure rate of non-union fracture healing. C2 C12 cells can be induced and differentiated into osteoblast under a certain condition. Although previous studies have demonstrated that bone morphogenetic protein 9（ BMP9） is highly capable of promoting osteogenic differentiation in C2C12 cells,the mechanism is largely unclear. We investigated the possible involvement and detailed role of Sonic hedgehog（ Shh）signaling in BMP9-induced osteogenic differentiation in C2 C12 cells. First, we constructed recombinant adenoviruses Ad-Shh,and then detected the expression of Shh by RT-PCR and Western blotting in C2 C12 cells. C2 C12 cells were infected with Ad-Shh in the presence of BMP9,the alkaline phosphatase（ ALP） activity was detected by quantitative and staining assay, calcium deposition was detected by Alizarin red S staining,the expression of osteopontin（ OPN）,osteocalcin（ OCN）,Id1,Id2,Dlx5 and Runx2 were detected by RT-PCR and /or Western blotting,the ectopic bone formation was detected by Micro-CT and H＆E staining. It was found that Ad-Shh could infectC2 C12 cells successfully. BMP9-induced early osteogenic marker,such as ALP activity,and late osteogenic markers,such as matrix mineralization,as well as the expression of OPN and OCN were enhanced by Ad-Shh. Furthermore,BMP9-induced expression of osteogenic pivotal transcription factors and ectopic bone formation were increased by Ad-Shh. Our results intensively revealed that enhancement of Shh signaling resulted in augmentation in BMP9-induced osteogenic differentiation in C2 C12 cells,implying that Shh signaling was involved and played a regulatory role in osteogenic differentiation in C2 C12 cells induced by BMP9.

关 键 词：Sonic HEDGEHOG信号通路 骨形态发生蛋白9 小鼠成肌细胞C2C12 成骨分化 

分 类 号：Q254[生物学—细胞生物学]