自噬在张力诱导终板软骨细胞退变过程中的变化

 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、3 h、12 h、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0 h组与3 h组为正常终板软骨细胞形态，呈多角形；12 h组略呈不规则形；24 h组和48 h组呈梭形改变。实时PCR显示24 h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24 h组和48 h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。

The expression and significance of autophagy in ICMT-induced endplate cartilage degeneration

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 cells 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-l 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 end-plate 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 h 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.