前期实验显示OA软骨细胞内提高低表达的Akt可促进软骨基质合成；同时观察到PLC-γ在OA软骨细胞内呈高表达。已知激活后PLC-γ可水解PIP3产生IP3与DAG参与调节细胞代谢，但PLC-γ对OA软骨细胞调控意义尚且不清。结合前人和我们的研究推测：①活化的PLC-γ可通过IP3/Ca2+与DAG/PKCδ启动MEK/ERK/Sp1及P70S6K/S6信号轴调控OA软骨基质代谢；② PLC-γ与Akt间具有密切关联的结构基础，并可能存在功能上的相互拮抗。本研究以体外培养的人正常/OA软骨细胞、HEK293T细胞及大鼠OA动物模型为研究对象，转染相关表达载体/药物处理，借助WB与RT-PCR等技术，检测PLC-γ调节基质代谢的信号调控网络；探讨Akt调控PLC-γ活性的机制；观察抑制PLC-γ活性对活体软骨损伤的影响，验证上述推测、阐明PLC-γ在OA病理进程中的意义以及预防和治疗OA的价值

Our previous study indicated that activated Phospholipase C-γ (PLC-γ) is higher in OA chondrocytes and activated Akt is lower in OA chondrocytes, compared with normal articular chondrocyte. Moreover, the increase of Akt expression caused by extracellular factors can promote extracellular matrix formation. PLC-γ is tyrosine phosphorylated by various growth factor receptors after binding of their cognate ligands. Tyrosine phosphorylation can turn protein structure into an enzymatically active form and generate second messengers by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol-trisphosphate (IP3). DAG subsequently activates protein kinase C(PKC ) and evokes phosphorylation of various cellular proteins. However, it is not clear to understand the role of PLC-γ in OA chondrocytes. Combined with other author’s studies on PLC-γ and Akt in other cell lines, we proposed that phosphorylation of PLC-γ1 at Tyr 783 and PLC-γ2 at Tyr759/Tyr1217 site is also the most crucial event in activating PLC-γ in articular chondrocyte as well as other cell lines, and the phosphorylation of PLC-γ at PLC-γ1-Ser 1248 and PLC-γ2-Ser677 site plays a negative role in PLC-γ activation. Moreover, Akt might interact with PLC-γ and result in the inhibition of PLC-γ activation by phosphorylating PLC-γ at PLC-γ1-Ser 1248 and PLC-γ2-Ser677 site. The increase of PLC-γ activation expression by suppressed-Akt activation in OA chondrocytes might trigger IP3/Ca2+ /MEK/ERK or P70S6K/S6 and DAG/PKCδ/MEK/ERK signaling axis, leading to the degradation of extracellular matrix in OA chondrocytes. In the present study, we will examine the protein and mRNA expression and distribution of activated PLC-γ and the activation of PLC-γ in normal and OA chondrocytes by western blot, the assay kit, and Real-time PCR, and analyze the difference. Meanwhile, the relevant signaling axis triggered by activation of PLC-γ will be monitored. We will also test the possibility that Akt regulates the activation of PLC-γ by binding to its SH3 domains and phosphorylating PLC-γ1-Ser 1248 and PLC-γ2-Ser677 site in normal and OA chondrocytes. The role of the inhibition of PLC-γ activation will be observed and analyzed in rat OA model. Eventually, the data will support the proposal that the activation of PLC-γ and the interaction with Akt lead to the degradation of extracellular matrix in OA chondrocytes, and it is helpful in understanding the role of PLC-γ in OA pathological progression and the prevention and therapy of OA.