Sirt6促进自噬抑制软骨细胞衰老的体外研究

Effects of Sirt6 on chondrocyte autophagy in chondrocyte senescence: an in vitro study

目的:明确Sirt6对软骨细胞衰老的影响,探讨自噬在Sirt6抑制软骨细胞衰老中的作用。方法:体外培养SD大鼠膝关节软骨细胞,通过连续传代致软骨细胞衰老,β-半乳糖苷酶(β-gal)染色检测细胞衰老,Western blot检测Sirt6的蛋白表达变化。原代软骨细胞中借助慢病毒敲低Sirt6或过表达Sirt6,比较各组的最大传代次数及细胞衰老情况,通过q PCR比较二型胶原(Collagen-Ⅱ)、蛋白聚糖(Aggrecan)、基质金属蛋白酶-13(MMP-13)及ADAMT-5的m RNA表达来探讨Sirt6对软骨细胞功能的影响。在软骨细胞中过表达Sirt6后通过免疫荧光染色观察细胞中自噬泡的变化,Western blot检测LC3-Ⅱ/LC3-Ⅰ的比例来探讨Sirt6对软骨细胞自噬的影响。在过表达Sirt6的同时通过使用自噬抑制剂3-甲基腺嘌呤(3-MA)来验证细胞自噬在Sirt6抑制软骨细胞衰老和功能退变中的作用。结果:连续体外培养软骨细胞,随着细胞传代次数的增加,Sirt6基因的表达量逐渐降低,原代软骨细胞中敲低Sirt6后,软骨细胞提前衰老,P16蛋白的表达量明显升高;软骨细胞中过表达Sirt6可以明显延缓细胞衰老;Sirt6可以促进软骨细胞自噬的发生;使用自噬抑制剂后可以明显抑制Sirt6对软骨细胞的保护作用。结论:提高Sirt6的表达水平可以延缓软骨细胞衰老及退变,且这种作用与Sirt6促进软骨细胞自噬相关。

Objective： To investigate the effects of Sirt6 in chondrocyte senescence and its effect on chondrocyte autophagy in chondrocyte senescence. Methods： The chondrocytes were isolated and cultured in vitro, and the chondrocyte senescence was induced by continuous passage. The chondrocyte senescence was detected by beta-galactosidase staining, and protein expression of Sirt6 was detected by Western blot. We then overexpressed or knocked down Sirt6 in chondrocytes using lentivirus, and compared the maximum number of passages and the percentages of senescent cells between different groups. The effects of Sirt6 on chondrocyte function were investigated by real-time PCR, to compare the m RNA expression of collagen-II, aggrecan, MMP-13, and ADAMTS-5 in each group. After overexpression of Sirt6 in chondrocytes, the presence of intracellular autophagosomes was analyzed by immunofl uorescence, and the ratio of LC3-II/LC3-I was analyzed by Western blot. In addition, the effect of autophagy on the inhibition of senescence and functional degeneration of the chondrocytes during overexpression of Sirt6 was verifi ed by using the autophagy inhibitor, 3-methyladenine. Results： Expression of Sirt6 decreased gradually with an increase in the number of cell generations, and expression of P16 increased significantly in chondrocytes with overexpressed Sirt6. Over expression of Sirt6 in chondrocytes signifi-cantly suppressed cellular senescence. Sirt6 promoted the autophagy of chondrocytes, and an autophagy inhibitor inhibited the protective effect of Sirt6 on chondrocytes. Conclusion： Increasing the expression level of Sirt6 candelay the senescence and degeneration of chondrocytes, and its effect is related to the promoted autophagy by Sirt6.