细胞自噬对饥饿环境下椎间盘髓核细胞的保护作用

目的: 探讨饥饿条件下椎间盘髓核细胞是否发生自噬及自噬对髓核细胞的作用。方法: 原代培养SD大鼠髓核细胞,对细胞表型进行鉴定后,将细胞分为正常对照组、3-甲基腺嘌呤(3-methyladenine,3-MA)加DMEM培养基组、3-MA加EBSS培养基组和EBSS饥饿组,接着用单丹磺酰戊二胺(monodansylcadaverine,MDC)染色、透射电镜和Western blotting观察各组细胞的自噬差异,最后采用CCK-8法测定细胞生长抑制率以及TUNEL法测定细胞凋亡率。结果: 成功培养出SD大鼠髓核细胞。与对照组相比,电镜和荧光显微镜下观察到EBSS培养基诱导的髓核细胞出现自噬囊泡,但是3-MA 抑制后细胞自噬泡明显减少;Western blotting检测发现LC3-II/LC3-I和Beclin-1/β-actin在EBSS处理组明显大于3-MA+EBSS处理组和对照组(P<0.05), 而3-MA+EBSS处理组的细胞抑制率和凋亡率明显高于EBSS处理组(P<0.05)。结论: 饥饿可以诱导大鼠椎间盘髓核细胞发生自噬,并且3-MA能抑制自噬发生。自噬对饥饿环境下的髓核细胞可能具有一定的保护作用。

Protective effect of autophagy on nucleus pulposus cells under starvation

AIM: To explore the possibility that the starvation environment induces autophagy of nucleus pulposus cells.METHODS: Primary rat nucleus pulposus cells was cultured and stained with toluidine blue,Alcian blue and immunocytochemistry for typeⅡ collagen.The cultured cells were divided into 4 groups: control group,3-methyladenine(3-MA)+DMEM group,3-MA+EBSS group and EBSS group.The cells were detected for autophay using monodansylcadaverine(MDC) staining,electron microscopy and Western blotting.At the same time,the inhibitory rate and apoptotic rate of the cells were detected by Cell Counting Kit-8(CCK-8) assay and TUNEL staining,respectively.RESULTS: Compared with control group,the autophagosomes were observed in nucleus pulposus cells under electron microscope and fluorescence microscope in EBSS group,and the 3-MA+EBSS treatment suppressed the formation of autophagosomes.The results of Western blotting analysis showed that the ratios of LC3-II/LC3-I and Beclin-1/β-actin in EBSS treatment group were higher than those in control group and 3-MA+EBSS treatment group.However,the apoptotic rate of nucleus pulposus cells and the inhibitory rate of cell viability were increased in 3-MA+EBSS treatment group.CONCLUSION: Autophagy of nucleus pulposus cells is induced by nutrient starvation,and 3-MA suppresses the response.Autophagy may have a protective effect on nucleus pulposus cells under the condition of starvation.