BMP9是目前诱导间充质干细胞（MSCs）成骨分化能力最强的BMPs成员之一。课题组研究表明：BMP9能诱导COX-2表达，且COX-2对BMP9诱导MSCs成骨分化有重要影响，但影响机制不清楚；BMP9能诱导PTEN表达，但PTEN抑制剂却增强BMP9诱导MSCs碱性磷酸酶（ALP）活性增加；COX-2沉默表达抑制BMP9诱导MSCs ALP活性增加，PTEN抑制剂可逆转此作用，但PGE2仅能部分逆转；BMP9合并COX-2过表达促进PTEN磷酸化，但BMP9合并COX-2沉默表达却明显抑制PTEN磷酸化。课题组推测：PTEN在BMP9诱导MSCs成骨分化中有重要作用，COX-2通过调节PTEN的功能平衡实现对BMP9作用的影响。本研究利用多种实验技术和方法，阐明PTEN在BMP9诱导MSC成骨分化过程中的作用；分析在BMP9诱导MSCs成骨分化中，COX-2调节PTEN水平的可能机制。

Our previous data has demonstrated that bone morphogenic protein 9 (BMP9) is one of the most potent member of BMPs to commit mesenchymal stem cells (MSCs) into osteo-progenitor lineage, although the detail mechanism remain unclear. BMP9 can induce the expression of cycloxygenase-2 (COX-2) in MSCs, and COX-2 can affects the osteogenesis induced by BMP9 greatly in MSCs. It has been reported that COX-2 is important for osteogenesis through the production of PGE2, which can activate PKA to produce more cAMP to promote the osteogenesis or bone fracture healing process by binding with EP2 and EP4 receptors. But, the mechanism of COX-2 affects the osteogenesis induced by BMP9 is unknown. Because PI3K/Akt is important for BMPs mediated osteogenesis and expression of COX-2, which can be negative regulated by PTEN, so we speculate that PTEN maybe involved in BMP9 induced osteogenesis in MSCs. With further investigation, we found the BMP9 can induce the expression of PTEN in MSCs, but PTEN specific inhibitor can enhance the ALP activity in MSCs induced by BMP9 with a concentration dependent fashion. Knockdown of COX-2 can reduce the ALP activity induced by BMP9 in MSCs, which can be fully reversed by PTEN inhibitor VO-OHpic, and can only be partly reversed by PGE2. This suggest that the effect of COX-2 on BMP9 induced osteogenesis is partly due to the production of PGE2, COX-2 may fulfill this function through other ways. With Western blot analysis, we found that over-expression of COX-2 combine with BMP9 can decrease the total protein level of PTEN, but increase the phosphorylation of PTEN, which can reduce the function of PTEN; knockdown of COX-2 combine with BMP9 can increase the total protein level of PTEN and decrease the phosphorylation of PTEN. These results indicate that PTEN is important for BMP9 to fulfill its function, and the effect of COX-2 on BMP9 induced osteogenesis in MSCs maybe result from the regulation of PTEN by COX-2. This proposal will follow a serial of well-established experiments to validate the importance of PTEN in the process of BMP9 induced osteogenesiss in MSCs, and analysis the possible mechanism of how COX-2 regulates the functional balance of PTEN in BMP9 induced osteogenesis in MSCs. With the practice of this proposal, we can expansion the knowledge of COX-2 in osteogenesis, and clarify the role of PTEN in BMP9 induced osteogenesis. It will accelerate the development of bone tissue engineering project, which will benefit the treatment or recover of bone fracture, bone defect and bone un-union diseases.