血清和营养剥夺增加椎间盘纤维环细胞的自噬通量：一项体外实验研究。
Serum and nutrient deprivation increase autophagic flux in intervertebral disc annulus fibrosus cells: an in vitro experimental study.
机构信息
Ferguson Laboratory for Orthopaedic and Spine Research, Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, University of Pittsburgh, 200 Lothrop Street, E1612 BSTWR, Pittsburgh, PA, 15213, USA.
Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan.
出版信息
Eur Spine J. 2019 May;28(5):993-1004. doi: 10.1007/s00586-019-05910-9. Epub 2019 Mar 7.
PURPOSE
The loss of nutrient supply is a suspected contributor of intervertebral disc degeneration. However, the extent to which low nutrition affects disc annulus fibrosus (AF) cells is unknown as nutrient deprivation has mainly been investigated in disc nucleus pulposus cells. Hence, an experimental study was designed to clarify the effects of limited nutrients on disc AF cell fate, including autophagy, the process by which cells recycle their own damaged components.
METHODS
Rabbit disc AF cells were cultured in different media with varying serum concentrations under 5% oxygen. Cellular responses to changes in serum and nutrient concentrations were determined by measuring proliferation and metabolic activity. Autophagic flux in AF cells was longitudinally monitored using imaging cytometry and Western blotting for LC3, HMGB1, and p62/SQSTM1. Apoptosis (TUNEL staining and cleaved caspase-3 immunodetection) and cellular senescence (senescence-associated β-galactosidase assay and p16/INK4A immunodetection) were measured.
RESULTS
Markers of apoptosis and senescence increased, while cell proliferation and metabolic activity decreased under the withdrawal of serum and of nutrients other than oxygen, confirming cellular stress. Time-dependent increases in autophagy markers, including LC3 puncta number per cell, LC3-II expression, and cytoplasmic HMGB1, were observed under conditions of reduced nutrition, while an autophagy substrate, p62/SQSTM1, decreased over time. Collectively, these findings suggest increased autophagic flux in disc AF cells under serum and nutrient deprivation.
CONCLUSION
Disc AF cells exhibit distinct responses to serum and nutrient deprivation. Cellular responses include cell death and quiescence in addition to reduced proliferation and metabolic activity, as well as activation of autophagy under conditions of nutritional stress. These slides can be retrieved under Electronic Supplementary Material.
目的
营养供应的丧失被怀疑是导致椎间盘退变的一个因素。然而，由于营养剥夺主要在椎间盘核髓细胞中进行研究，因此，低营养对椎间盘纤维环（AF）细胞的影响程度尚不清楚。因此，设计了一项实验研究，以阐明有限营养对椎间盘 AF 细胞命运的影响，包括自噬，这是细胞回收自身受损成分的过程。
方法
将兔椎间盘 AF 细胞在不同血清浓度的不同培养基中于 5%氧气下培养。通过测量增殖和代谢活性来确定血清和营养浓度变化对细胞的反应。使用成像细胞术和 LC3、HMGB1 和 p62/SQSTM1 的 Western blot 纵向监测 AF 细胞中的自噬通量。通过 TUNEL 染色和 cleaved caspase-3 免疫检测测量细胞凋亡（凋亡），通过衰老相关β-半乳糖苷酶测定和 p16/INK4A 免疫检测测量细胞衰老。
结果
在血清和除氧气以外的营养物质去除下，细胞增殖和代谢活性下降，同时凋亡和衰老的标志物增加，证实了细胞应激。在营养减少的情况下，观察到自噬标志物包括细胞内 LC3 斑点数/细胞、LC3-II 表达和细胞质 HMGB1 的时间依赖性增加，而自噬底物 p62/SQSTM1 随时间减少。总的来说，这些发现表明在血清和营养剥夺下，椎间盘 AF 细胞中自噬通量增加。
结论
椎间盘 AF 细胞对血清和营养剥夺有明显的反应。细胞反应除了增殖和代谢活性降低外，还包括细胞死亡和静止，以及在营养应激下自噬的激活。这些幻灯片可以在电子补充材料中检索到。
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