AP2α在BMP9诱导C3H10成骨分化中的作用及机制探讨

Effect of activator protein 2α on BMP9-induced osteogenesis differentiation of C3H10 and its mechanism

摘要:

目的：研究转录蛋白2（activator protein 2，AP2）α在骨形态发生蛋白（bone morphogenetic protein，BMP）9诱导小鼠间充质干细胞C3H10成骨分化中的作用及可能的分子机制。方法：重组腺病毒 Ad-BMP9感染小鼠C3H10细胞，显性负性突变体AP2α-△bHLH和AP2α-△TAD抑制 AP2α转录活性，通过RT-PCR、real-time PCR检测AP2α、BMP9及成骨相关基因的表达水平，Western blot检测骨桥蛋白（osteopontin，OPN）表达，碱性磷酸酶（alkaline phosphatase，ALP）活性测定、茜红素染色实验观测C3H10成骨分化。结果：BMP9具有强促成骨分化的作用，对AP2α的表达水平无影响，AP2α显性负性突变体可抑制BMP9诱导的成骨基因骨钙蛋白（osteocalcin，OC）、OPN及骨保护素（osteoprotegerin，OPG）表达的增高（OC：F=56.929，P=0.000；OPN：F=54.789，P=0.000；OPG：F=159.451，P=0.000），ALP活性（F=69.776，P=0.000）及红色钙盐沉积减少。结论：BMP9可能通过影响AP2α的转录活性调控成骨分化进程。

Abstract:

Objective：To research the effect of activator protein 2α on the process of bone morphogenetic protein 9（BMP9）-induced osteogenesis differentiation of mouse mesenchymal stem cells（C3H10） and its mechanism. Methods：C3H10 cells were infected with Ad-BMP9. Adenovirus-mediated dominant negative mutants Ad-dnAP2α-△bHLH and Ad-dnAP2α-△TAD were used to inhibit AP2α transcriptional activity. RT-PCR，real-time PCR and Western blot methods were used to detect the expression of AP2α，BMP9 and osteogenetic related markers. Alkaline phosphatase reading/staining and alizarin red staining were performed to detect the status of osteogenetic differentiation. Results：Over-expression of BMP9 could accelerate osteogenesis differentiation but did not affect the expression of AP2α. AP2α dominant negative mutants could inhibit the BMP9-induced increase of osteogenesis related markers osteocalcin（OC），osteopontin（OPN）and osteoprotegerin（OPG）expression（OC：F=56.929，P=0.000；OPN：F=54.789，P=0.000；OPG：F=159.451，P=0.000）and reduce the alkaline phosphatase activity（F=69.776，P=0.000） and red staining of calcium salt deposi－tion. Conclusion：AP2α transcription activity might regulate the process of BMP9-induced osteogenesis differentiation.