周期性应力下胰岛素样生长因子1受体对大鼠软骨细胞功能的影响

目的探讨周期性机械应力条件下胰岛素样生长因子1型受体(IGF1R)对大鼠软骨细胞增殖及细胞外基质合成的影响。 方法体外分离培养大鼠软骨细胞，随机分为4组：加压0 h组、加压0.5 h组、加压1 h组、加压2 h组，采用Western Blot法检测各组细胞IGF1R的表达及磷酸化水平，并在此基础上将同代无处理的软骨细胞在加压各组检测的同时给予同样检测即为静态组，应用Gel-Pro Analyzer软件进行半定量灰度分析比较各时间段静态和加压两两各组磷酸化IGF1R/总IGF1R水平差异。另取大鼠软骨细胞随机分为静态组、加压对照组、IGF1R阻断组、加压后IGF1R阻断组，IGF1R阻断采用顺式－3－[8－胺基－1－(2－苯基－喹啉－7－基)－咪唑并[1，5－a]吡嗪－3－基]－1－甲基－环丁醇(OSI－906)或者以IGF1R shRNA两种方式。阻断IGF1R 1 h后Western Blot法检测各组细胞磷酸化细胞外信号调节激酶1/2(ERK 1/2)表达及磷酸化水平；8 h后实时荧光定量PCR检测各组2型胶原(collagen Ⅱ)、蛋白聚糖(aggrecan)表达；3 d后采用直接细胞计数方式及细胞计数试剂盒(CCK-8)对软骨细胞增殖状况进行测定。应用SPSS 18.0软件进行相关统计学分析，两组间差异采用t检验比较。 结果磷酸化IGF1R/总IGF1R蛋白水平在加压0 h组与静态组之间差异无统计学意义(t＝0.255, P＝0.811)，而在0.5 h组，1 h组，2 h组较相对应的静态组增高，差异具有统计学意义(t＝－5.881、－6.172、－10.518，P均小于0.05)。IGF1R被OSI-906或shRNA抑制后，加压处理的ERK 1/2磷酸化水平显著降低(OSI-906阻断后t＝3.074，shRNA阻断后t＝3.990，P均小于0.05)，细胞外基质collagen Ⅱ(OSI-906阻断后t＝3.243, shRNA阻断后t＝3.621，均为P<0.05)、aggrecan(OSI-906阻断后t＝3.128，shRNA阻断后t＝3.608，P均小于0.05)基因表达水平显著降低，大鼠软骨细胞增殖能力减弱，差异均具有统计学意义(OSI-906阻断后t＝2.835、shRNA阻断后t＝3.467，均为P<0.05)。 结论周期性机械应力通过压力感受器IGF1R将机械信号转变为生物化学信号，激活ERK 1/2信号通路促进软骨细胞增殖和细胞外基质合成。

Effects of insulin-like growth factor 1 receptor on rat chondrocytes under cyclic mechanical stress

ObjectiveTo investigate the effects of insulin-like growth factor 1 receptor (IGF1R) on proliferation and extracellular matrix synthesis in rat chondrocytes under cyclic mechanical stress. MethodsThe rat chondrocytes were isolated and cultured in vitro. They were randomly divided into four groups: 0 h stress group, 0.5 h stress group, 1 h stress group, and 2 h stress group; each group were treated by cyclic mechanical stretch stress treated for 0 h, 0.5 h, 1 h and 2 h respectively. Western Blot analysis was performed for IGF1R and phospho-IGF1R levels and chose an array of non-pressure cells as control of each group. The Gel-Pro Analyzer software was used for semi-quantitative analysis of gray value to compare the differences of phospho-IGF1R/total IGF1R between non-pressure and pressure groups in each time periods. The blank group and cyclic mechanical stretch treated cells were separately divided into control group and IGF1R-suppressor group which treated with Linsitinib or IGF1R shRNA. The expression of extracellular regulated protein kinases (ERK)1/2 and phospho-ERK1/2 were determined by Western blot 1 h later. After 8 h, collagen Ⅱ and aggrecan levels were analyzed by qPCR. Three days later, cell counting or CCK-8 assays were performed to examine the cell proliferation of each group.SPSS 18.0 software was used for statistical analysis, and student’s t test was used to compare the differences between the two groups. ResultsIn 0 h stress groups, the phospho-IGF1R levels had no difference(t=0.255, P=0.811). The levels of phospho-IGF1R were significantly increased in the cyclic mechanical stretch treated groups(t=-5.881, -6.172 , -10.518, all P<0.05). The inhibitors of IGF1R obviously decreased the phospho-ERK1/2 expression(OSI-906 suppressor group t=3.074, IGF1R shRNA suppressor group t=3.990, all P<0.05). The mRNA levels of collagen Ⅱ(OSI-906 suppressor group t=3.243, IGF1R shRNA suppressor group t=3.621, both P <0.05) and aggrecan (OSI-906 suppressor group t=3.128, IGF1R shRNA suppressor group t=3.608, both P <0.05)significantly reduced thus extracellular matrix synthesis was blocked because of IGF1R suppressors.The IGF1R inhibitors significantly contributed to the attenuation of cyclic mechanical stretch treated cells proliferation(OSI-906 suppressor group t=2.835, IGF1R shRNA suppressor group: t=3.467, all P <0.05). ConclusionCyclic mechanical stretch converts mechanical signals into biochemical signals through the pressure receptor IGF1R and activates the ERK1/2 signaling pathway to promote chondrocyte proliferation and extracellular matrix synthesis.