Ihh-PTHrP信号轴介导髁突软骨细胞对压力微环境改变的调控研究

目的 探究Ihh-PTHrP负反馈信号通路与静压力下髁突软骨细胞生物学响应的关系。方法 体外分离培养4周龄健康雌性新西兰大白兔髁突软骨细胞，100 kPa静压力条件下，分别对P2代髁突软骨细胞进行0、1、2、3、4 h的加压处理；采用Western印迹的方法比较分析髁突软骨细胞COL2A1、Ihh和PTHrP的蛋白表达变化；通过RT-qPCR检测分析静压力对Ihh和PTHrP基因水平表达的影响；选取0 h和3 h组，使用扫描电子显微镜扫描分析静压力对髁突软骨细胞形态的影响；采用SPSS 19.0软件包对数据进行统计学分析。结果 Western印迹实验发现：压力加载3 h后，COL2A1出现明显高表达；Ihh蛋白表达在0～2 h内逐渐升高，在2～4 h内却逐渐降低；PTHrP在0～2 h内蛋白表达逐渐升高，并在2～4 h内维持着一个较高水平的表达。RT-qPCR检测发现Ihh在压力加载1 h时出现明显高表达，而PTHrP则在2 h时出现明显高表达。100 kPa静压力加载3 h后，髁突软骨细胞突起增多、变长。结论 兔髁突软骨细胞对压力微环境的改变具有一定的适应能力；适宜的静压力可以提高髁突软骨细胞的成软骨能力；Ihh-PTHrP负反馈信号通路参与髁突软骨细胞对压力微环境改变的适应性改建过程。

Effects of static pressure on condylar chondrocytes mediated by Ihh-PTHrP pathway in rabbit temporomandibular

Objective　　To explore the relationship between Ihh PTHrP feedback pathway and the biological response to alteration of pressure microenvironment in the condylar chondrocytes.Methods　　The condylar chondrocytes from the temporomandibular joint of 4 week old New Zealand white rabbit were aseptically dissected and cultured in vitro.The monolayer of condylar chondrocytes was subjected to the static pressure of 100 kPa at different times (0 h,1 h,2 h,3 h and 4 h).Production of COL2A1,Ihh and PTHrP was examined by western blot and quantitative real time PCR.The changes of condylar chondrocytes (100 kPa,3 h) morphology were analyzed by scanning electron microscope compared with the control group (100 kPa,0 h).One way ANOVA was performed using SPSS19.0 software package for statistical analysis.Results　　The western blot results showed that the exposure of chondrocytes to 100 kPa resulted in significant up regulation of COL2A1 after 3h.Increased expression of Ihh and PTHrP could be found in the condylar chondrocytes in respond to static pressure within 0～2h.Moreover,Ihh reduced in the condylar chondrocytes within 2～4h,whereas the expression of PTHrP maintained a high level after 2h′s treatment.The gene expression of Ihh and PTHrP was similar to the proteins.The result of scanning electron microscope showed that the process of the condylar chondrocytes prolonged after 3 hours treatment.Conclusions　　The condylar chondrocytes is good at adapting to the alteration of pressure microenvironment.It is sure that a suitable static pressure loading can accelerate chondrogenesis of condylar chondrocytes.Ihh PTHrP feedback pathway is involved in the regulation of the alteration of pressure microenvironment in condylar chondrocytes.