RhoA/ROCK信号通路在终板软骨细胞体外自然退变中的变化

Change and Significance of RhoA/ROCK signaling pathway in the model with natural degeneration of the rat endplate chondrocytes

目的 探讨RhoA/ROCK信号通路在终板软骨细胞体外自然退变中的表达变化.方法 提取大鼠终板软骨细胞并建立体外的终板软骨细胞自然退变模型,依次选取P2代细胞（空白对照组）至P5代细胞（自然退变组）,以及治疗组（P5代细胞加入ROCK信号通路抑制剂Y27632组）,倒置显微镜观察细胞形态,利用甲苯胺蓝染色对软骨细胞进行观察及表型鉴定.激光共聚焦显微镜检测空白对照组和自然退变组的细胞骨架变化.RT-PCR方法检测各组细胞中Ⅱ型胶原、SOX9、蛋白聚糖基因及信号通路中ROCK-1、ROCK-2基因的表达变化;免疫印迹检测ROCK-1、ROCK-2蛋白表达变化及利用试剂盒检测RhoA蛋白活性表达变化.结果 随着体外自然传代的发生终板软骨细胞表型基因表达逐渐丢失;细胞骨架在激光共聚焦显微镜观察下可见P5代终板软骨细胞较P2代细胞骨架明显变细.自然退变组（P5代）终板软骨细胞较空白对照组（P2代细胞）Ⅱ型胶原（P5/P2=0.248,P＜0.001）、蛋白多糖（P5/P2=0.172,P＜0.001）及SOX9（P5/P2 =0.499,P＜0.001）表达明显降低,RhoA/ROCK信号通路中关键基因ROCK-1（P5/P2=0.625,P＜0.001）表达降低、ROCK-2（PS/P2=2.527,P＜0.001）表达升高;而活性蛋白检测发现活性RhoA的量P5代较P2代明显增加.治疗组（P5代细胞中加入ROCK抑制剂Y27632）中抑制ROCK信号以后ROCK-1、ROCK-2表达较P5代细胞均明显降低,蛋白亦是如此.而治疗组中Ⅱ型胶原、蛋白聚糖、SOX9等基因表达则明显回升.结论 细胞体外自然传代从P2代细胞至P5代时,终板软骨细胞发生明显退变.并提示可通过调控RhoA/ROCK信号通路来保护终板软骨细胞退变.

Objective To explore the change and Significance of RhoA/ROCK signaling pathway in the model with natural degeneration of the rat endplate chondrocytes.Methods 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.Results 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 （PS/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.Conclusion 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.