CAJ | 학술논문

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。
목적：탐토Wnt/β-련단백（ catenin ）신호통로중관건기인재종판연골세포자연퇴변중적표체변화급의의。방법분리배양대서종판연골세포，건립종판연골세포체외자연전대퇴변모형。분위공백대조조（ P2대세포）、자연전대조（ P5대세포）급wnt신호통로억제조，도치현미경관찰세포형태，HE염색급갑분알람염색검측종판연골세포표형개변。실시취합매련반응검측각조세포중Ⅱ형효원、단백다당、SOX-9기인급β-catenin적표체변화；면역인적검측β-catenin단백표체변화。격광공취초현미경검측β-catenin재세포내적표체여정위。결과수착자연전대적진행종판연골세포표형축점주실；P5대종판연골세포교P2대세포Ⅱ형효원（P5/P2＝0.11，P＝0.0039）、단백다당（P5/P2＝0.32，P＝0.0046）급SOX-9（P5/P2＝0.58，P＝0.0168）표체명현강저，이Wnt/β-catenin신호통로중관건기인β-catenin（P5/P2＝1.62，P＝0.0082）표체명현증고；억제wnt 신호통로후β-catenin （XAV-939/P5＝0.23，P＝0.0017）표체강저，Ⅱ형효원（XAV-939/P5＝2.60，P＝0.0180）、단백다당（XAV-939/P5＝2.56，P＝0.0041）급SOX-9（XAV-939/P5＝1.47，P＝0.0382）표체증고。격광공취초현미경관찰가견P5대종판연골세포교P2대중β-catenin표체증고차명현입핵，억제wnt신호통로후β-catenin표체명현감소。결론β-catenin기인재종판판연골세포체외퇴변과정중구유중요적작용，조공Wnt/β-catenin신호통로유가능보호종판연골적퇴변。
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.