Wnt／β-catenin 信号通路在终板软骨细胞体外自然退变模型中的表达  

Expression and significance of Wnt/β-catenin signaling pathway in vitro natural degeneration model of endplate chondrocytes in rats

作　　者：郑权[1] 徐宏光[1] 张晓玲[2] 王弘[1] 汪景[2] 王传东[2] 宋俊兴 熊寿良[1] 赵泉来[1] 

机构地区：[1]皖南医学院第一附属医院弋矶山医院脊柱外科,安徽省芜湖市241001 [2]中国科学院上海生命科学院,上海交通大学健康科学研究所骨科细胞与分子生物学实验室 [3]芜湖市中医院骨七科

出　　处：《中华医学杂志》2014年第31期2464-2467,共4页National Medical Journal of China

基　　金：国家自然科学基金(81272048、30973025、81311130314)；卫生部公益性行业专项基金(201002018)；安徽省自然科学基金

摘　　要：目的：探讨Wnt/β-联蛋白（ catenin ）信号通路中关键基因在终板软骨细胞自然退变中的表达变化及意义。方法分离培养大鼠终板软骨细胞，建立终板软骨细胞体外自然传代退变模型。分为空白对照组（ P2代细胞）、自然传代组（ P5代细胞）及wnt信号通路抑制组，倒置显微镜观察细胞形态，HE染色及甲苯胺蓝染色检测终板软骨细胞表型改变。实时聚合酶链反应检测各组细胞中Ⅱ型胶原、蛋白多糖、SOX-9基因及β-catenin的表达变化；免疫印迹检测β-catenin蛋白表达变化。激光共聚焦显微镜检测β-catenin在细胞内的表达与定位。结果随着自然传代的进行终板软骨细胞表型逐渐丢失；P5代终板软骨细胞较P2代细胞Ⅱ型胶原（P5/P2＝0.11，P＝0.0039）、蛋白多糖（P5/P2＝0.32，P＝0.0046）及SOX-9（P5/P2＝0.58，P＝0.0168）表达明显降低，而Wnt/β-catenin信号通路中关键基因β-catenin（P5/P2＝1.62，P＝0.0082）表达明显增高；抑制wnt 信号通路后β-catenin （XAV-939/P5＝0.23，P＝0.0017）表达降低，Ⅱ型胶原（XAV-939/P5＝2.60，P＝0.0180）、蛋白多糖（XAV-939/P5＝2.56，P＝0.0041）及SOX-9（XAV-939/P5＝1.47，P＝0.0382）表达增高。激光共聚焦显微镜观察可见P5代终板软骨细胞较P2代中β-catenin表达增高且明显入核，抑制wnt信号通路后β-catenin表达明显减少。结论β-catenin基因在终板板软骨细胞体外退变过程中具有重要的作用，调控Wnt/β-catenin信号通路有可能保护终板软骨的退变。Objective To explore the expression and significance of Wnt /β-catenin signaling pathway in the natural degeneration of endplate chondrocytes in rats.Methods Endplate chondrocytes extracted from lumbar vertebrae were divided into control （P2 cell）, naturally passaged （P5 cell） and wnt signaling pathway inhibition groups.The morphology of endplate chondrocytes was observed by inverted microscope.Hematoxylin and eosin （ HE） and toluidine blue stains were used to identify their phenotypes.TypeⅡcollagen marker , SOX-9 and aggrecan genes were detected by reverse transcription-polymerase chain reaction （ RT-PCR） to verify the degeneration model.Based on this model , the changes of β-catenin were detected by RT-PCR and Western blot.Laser confocal microscopy was used to detect the expression and localization of β-catenin within endplate chondrocytes.Results With natural passaging , endplate cartilage cells appeared spindle-shaped and gradually lost chondrocytic phenotypes.The levels of type Ⅱ collagen （P5/P2=0.11, P=0.003 9）, SOX-9 （P5/P2=0.58, P=0.016 8） and aggrecan （P5/P2=0.32, P=0.004 6） significantly reduced;β-catenin （P5/P2=1.62, P=0.008 2） significantly increased.β-catenin was down-regulated （XAV-939/P5=0.23, P=0.001 7） in inhibition group.And type Ⅱcollagen （XAV-939/P5=2.60, P=0.018 0）, SOX-9 （XAV-939/P5 =1.47, P=0.038 2） and aggrecan （XAV-939/P5=2.56, P=0.004 1） significantly increased.β-catenin had a higher expression and obviously entered into nuclear transcription in P5 generation and decreased in inhibition group.Conclusion β-catenin plays an important role in the in vitro degeneration of endplate chondrocytes.There are great potentials for protecting endplate cartilage degeneration by regulating the Wnt /β-catenin signaling pathway.

关 键 词：椎间盘 软骨细胞 信号传递 细胞衰老 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]