CAJ | 학술논문

目的探讨自噬在张力诱导终板软骨细胞退变过程中的变化及作用.方法取10只清洁级SD大鼠腰椎终板软骨进行细胞培养.对P1代终板软骨细胞分别加载间歇循环张力(10％伸长率,0.5 Hz)0 h、3h、12h、24 h、48 h.以倒置相差显微镜观察细胞形态学变化,实时PCR与蛋白印迹法检测软骨标志基因Ⅱ型胶原、转录因子SOX-9及蛋白多糖转录因子、Beclin-1和LC3基因表达的变化,以单丹磺酰戊二胺染色观察自噬小体.MTT(3-2,5-二苯基四氮唑溴盐染色)法检测3-甲基腺嘌呤(自噬抑制剂)刺激前后的细胞存活率.结果间歇循环张力诱导后0h组与3h组为正常终板软骨细胞形态,呈多角形；12h组略呈不规则形；24h组和48 h组呈梭形改变.实时PCR显示24h组和48 h组中Ⅱ型胶原、转录因子SOX-9及蛋白多糖的表达量降低；自噬相关基因LC3和Beclin-1表达量呈时间依赖性增加.单丹磺酰戊二胺染色显示24h组和48h组自噬发生率呈时间依赖性增加.MTT结果显示细胞存活率呈降低趋势；添加3-甲基腺嘌呤刺激后细胞活性减弱、存活率降低.结论间歇循环张力刺激下终板软骨细胞表型逐渐丧失；自噬相关基因LC3与Beclin-1表达明显上调,但细胞活性降低；抑制自噬水平可降低细胞存活率,提示自噬参与了间歇循环张力诱导的终板软骨细胞退变过程.
목적 탐토자서재장력유도종판연골세포퇴변과정중적변화급작용.방법 취10지청길급SD대서요추종판연골진행세포배양.대P1대종판연골세포분별가재간헐순배장력(10％신장솔,0.5 Hz)0 h、3h、12h、24 h、48 h.이도치상차현미경관찰세포형태학변화,실시PCR여단백인적법검측연골표지기인Ⅱ형효원、전록인자SOX-9급단백다당전록인자、Beclin-1화LC3기인표체적변화,이단단광선무이알염색관찰자서소체.MTT(3-2,5-이분기사담서추염염색)법검측3-갑기선표령(자서억제제)자격전후적세포존활솔.결과 간헐순배장력유도후0h조여3h조위정상종판연골세포형태,정다각형；12h조략정불규칙형；24h조화48 h조정사형개변.실시PCR현시24h조화48 h조중Ⅱ형효원、전록인자SOX-9급단백다당적표체량강저；자서상관기인LC3화Beclin-1표체량정시간의뢰성증가.단단광선무이알염색현시24h조화48h조자서발생솔정시간의뢰성증가.MTT결과현시세포존활솔정강저추세；첨가3-갑기선표령자격후세포활성감약、존활솔강저.결론 간헐순배장력자격하종판연골세포표형축점상실；자서상관기인LC3여Beclin-1표체명현상조,단세포활성강저；억제자서수평가강저세포존활솔,제시자서삼여료간헐순배장력유도적종판연골세포퇴변과정.
Objective To investigate the relationship between autophagy and the endplate chondrocytes degeneration with intermittent cyclic mechanical tension (ICMT) in vitro.Methods The primary lumbar endplate cartilage ceils of 10 SD rats were cultured and the P1 generation was selected to treat with ICMT (10％,0.5 Hz) for 0 h,3 h,12 h,24 h,48 h.The cell morphology changes were observed through inverted phase contrast microscope.The expression of cartilage marker gene type Ⅱ collagen,SOX-9 and proteoglycan expression were observed by RT-PCR.Beclin-1 and LC3 expression of end-plate chondrocytes were detected by RT-PCR and Western blotting.The monodansylcadaverine (MDC) staining was used to detect autophagy rate in the endplate chondrocytes.The cells activity was detected by MTT assay.Results After ICMT loading,the cells of 0 h group and 3 h group kept normal morphology,but the cells of 12 h group presented irregular shape.The cells of 24 b group and 48 h group tended to assume a spindle-shaped morphology.The expression of type Ⅱ collagen,proteoglycan and Sox-9 in 24 h group and 48 h group decreased obviously.The results of RT-PCR and Western blotting showed that autophagy-related genes LC3 and Beclin-1 expression in time dependent increasing.The results of MDC staining showed that the occurrence of autophagy were significantly increased in 24 h and 48 h groups after ICMT,but MTT analysis showed the cells viability was significantly decreased,and the cell viability was significantly decreased with the stimulation of 3-methyl-adenine.Conclusion After ICMT loading,the endplate chondrocytes will lose their phenotype gradually.The expression of autophagy-related genes LC3 and Beclin-1 are significantly increased,but the cell viability is significantly decreased.Inhibiting the autophagy can reduce the cell survival rate.Autophagy may participate in the endplate chondrocytes degeneration process induced by ICMT.