Ma Mingming, Xu Hongguang, Zhang Xiaoling, Wang Hong, Zheng Quan, Xu Jiajia, Shen Xiang, Zhang Shufeng
Department of Orthopedic Surgery, Yijishan Hosptial, Wannan Medical College, Wuhu 241001, China.
Email:
Zhonghua Yi Xue Za Zhi. 2015 Nov 3;95(41):3373-7.
To explore the change and Significance of RhoA/ROCK signaling pathway in the model with natural degeneration of the rat endplate chondrocytes.
Endplate chondrocytes were selected by enzyme digestion and cultured in vitro to divided into control (P2 cells), naturally passaged (P5 cells) groups and treatment group (P5+ROCK Inhibitor Y27632). The phenotype of endplate chondrocytes were identified by toluidine blue stains and F-actin stains. Type II collagen, aggrecan and SOX9 genes were examed by Real-time RT-PCR to verify the degeneration model. The RhoA/ROCK signaling pathway related gene ROCK-1, ROCK-2 were detected by RT-PCR and Western blot. The actived RhoA was examed by active-RhoA detection and Western blot.
With the passaging,endplate chondrocytes completely lost the original cell morphology, the levels of type II collagen (P5/P2=0.248, P<0.001), aggrecan (P5/P2=0.172, P<0.001) and SOX9 (P5/P2 =0.499, P<0.001) significantly reduced. There is also a certain reduction of ROCK-1 (P5/P2=0.652, P<0.001), but ROCK-2 (P5/P2=2.527, P<0.001) expression increased significantly. And the active-RhoA were Significant increased too.ROCK-1 AND ROCK-2 were down-regulated in the treatment group. And type II collagen, aggrecan, SOX9 significantly increased.
The degeneration of endplate chondrocytes with decreased ROCK-1 expression but increased active-RhoA and ROCK-2 expression suggest that RhoA/ROCK signaling pathway play an important role in the in vitro degeneration of endplate chondrocytes.Modulating the expression of RhoA/ROCK signaling pathway may be a new method of solving the problem of the degeneration of intervertebral disc.
探讨RhoA/ROCK信号通路在大鼠终板软骨细胞自然退变模型中的变化及意义。
采用酶消化法选取终板软骨细胞并进行体外培养，分为对照组（P2代细胞）、自然传代组（P5代细胞）和处理组（P5代+ROCK抑制剂Y27632）。通过甲苯胺蓝染色和F-肌动蛋白染色鉴定终板软骨细胞的表型。采用实时逆转录聚合酶链反应（Real-time RT-PCR）检测Ⅱ型胶原、聚集蛋白聚糖和SOX9基因，以验证退变模型。通过逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测RhoA/ROCK信号通路相关基因ROCK-1、ROCK-2。采用活性RhoA检测和蛋白质免疫印迹法检测活化的RhoA。
随着传代，终板软骨细胞完全丧失原有的细胞形态，Ⅱ型胶原（P5/P2=0.248，P<0.001）、聚集蛋白聚糖（P5/P2=0.172，P<0.001）和SOX9（P5/P2 =0.499，P<0.001）水平显著降低。ROCK-1也有一定程度降低（P5/P2=0.652，P<0.001），但ROCK-2表达显著增加（P5/P2=2.527，P<0.001）。活化的RhoA也显著增加。处理组中ROCK-1和ROCK-2表达下调，Ⅱ型胶原、聚集蛋白聚糖、SOX9显著增加。
终板软骨细胞退变时ROCK-1表达降低，但活化的RhoA和ROCK-2表达增加，提示RhoA/ROCK信号通路在终板软骨细胞体外退变中起重要作用。调节RhoA/ROCK信号通路的表达可能是解决椎间盘退变问题的新方法。
