Sonic Hedgehog串话人胰岛素样生长因子-1介导的信号通路促进髁突肥大软骨过生长的机制

Mechanism of Cooperation Between Sonic Hedgehog and Insulin-Like Growth Factor-1 in Promoting Condylar Hyperplasia Cartilage Overgrowth

目的：探讨Sonic hedgehog（Shh）与人胰岛素样生长因子-1（IGF-1）介导的信号通路在促进髁突肥大病理进程中的作用及串话机制。方法：选取正常髁突及髁突肥大软骨标本,以及骨髓间充质细胞（BMSC）,体外分离进行平板及三维培养,按不同的实验目的划分为不同的比较组,每个比较组按不同的实验目的各含不同的刺激组,分别添加IGF-1,NVP-AEW541,Cyclpamine,U0126/LY294002因子等单独或联合刺激,孵育后收获细胞进行计数或进行real-time PCR或Western Blot实验。结果：髁突肥大软骨细胞中加入Cyclopamine,COLX、PCNA、MMP-13基因和蛋白表达量明显降低,而cleaved-caspase-3明显升高（P〈0.05）,细胞数目明显降低（P〈0.05）。添加Cyclopamine刺激的髁突肥大软骨细胞,磷酸化状态P-AKT和P-ERK均显著降低（P〈0.05）,Cleavedcaspase-3蛋白表达水平显著升高（P〈0.05）。在BMSC加强IGF-1及Shh的刺激后COLX、COL2、PCNA、MMP-13、BCL2基因的表达量均明显升高（P〈0.05）。单独加强IGF-1或Shh刺激,均可引起另一信号通路的加强（P〈0.05）。结论：Shh与IGF-1介导的信号通路存在串话机制促进肥大髁突软骨细胞及间充质细胞增殖、分化及凋亡的作用,并通过PI3-K AKT和MAPK-ERK通路发挥作用。

Objective：To investigate the crosstalk between Sonic Hedgehog（Shh）pathway and insulinlike growth factor-1（IGF-1）pathway and its regulating effects in condylar hyperlplasia（CH）chondrocytes.Methods：Chondrocytes were isolated from CH and normal cartilage（NC）specimens,and cultured in alginate beads or monolayer,then were treated with IGF-1 or specific in-hibitors such as Cyclopamine,NVP-AEW541,U0126,and LY294002.Thereafter,gene and protein expression levels of IGF-1,IGF-1 receptor（IGF-1 R）,collagen typeⅡ（COL2）,typeΧwere evaluated by realtime PCR and Western blotting.Results：The expressions of specific genes and proteins relate to cellular proliferation,differentiation and anti-apoptosis,such as COLX,COL2,PCNA,MMP-13,BCL2 were suppressed by the addition of Cyclopamine in CH chondrocytes and BMSC.Reversely,genes and proteins relatea to apoptosis were elevated.The crosstalk between Shh pathway and IGF-1 pathway was existed in CH chondrocytes,which promoted human TMJ cartilage overgrowth via MAPK-ERK and PI3-K AKT pathway CH chondrocytes significantly enhanced mRNA and protein expressions of IGF-1 and IGF-1 R,as compared with NC chondrocytes.Furthermore,enriched IGF-1 enhanced CH chondrocytes proliferation,up-regulated COL2 A1 expression.Besides,IGF-1-mediated CH chondrocytes proliferation mainly depended on the MAPK-ERK pathway.Conclusion：The crosstalk between Shh pathway and IGF-1 pathway promotes human TMJ cartilage overgrowth in the developing process of CH by via MAPK-ERK and PI3-K AKT pathway.