自噬参与压力诱导兔髓核细胞退变的研究

Involvement of autophagy in the progression of compression-induced nucleus pulposus cell degener-ation and death

目的观察自噬是否参与压力诱导兔髓核细胞退变，探讨压力诱导髓核细胞退变机制。方法从3月龄兔胸腰段脊柱提取兔髓核细胞培养，取第2代兔髓核细胞，1Mpa压力环境下培养12、24、48h。细胞活力与细胞毒性检测观察压力对细胞生长的影响，透射电镜观察压力条件下细胞的微观结构。单丹磺酰尸胺（MDC）染色观察细胞内自噬性空泡结构及自噬率，实时荧光定量聚合酶链反应（FQ—PCR）观察自噬相关基因在压力条件下的表达。结果压力导致细胞衰老及死亡，透射电镜见细胞内存在自噬体结构，MDC染色后行流式细胞学检测显示12、24、48h自噬率分别为（1．580±0．171）％、（7．930±0．252）％、（13．530±1．206）％，PER结果显示压力诱导自噬相关基因（LC3B、Beclin-1）表达量与正常条件下表达明显增多（P〈0．05），且随着压力培养时间延长，LC3B、Beclin-1表达量也明显增多（P〈0．05）。结论自噬参与压力诱导细胞退变的过程，为压力诱导椎间盘髓核细胞退变的防治提供新的途径。

Objective To investigate the involvement of autophagy in the progression of compres- sion-induced intervertebral disc （IVD） degeneration. Methods Rabbit nucleus pulposus （NP） cells were isolated from the thoracolumbar IVD of 3-month-old Japanese white rabbits. The rabbit NP cells at the sec- ond generation were cultured under the compression condition of 1 Mpa for 12, 24 or 48 h. Cell viability was determined by using cell counting Kit-8 （CCK8）. The uhrastructural features of rabbit NP cells ex- posed to compression were examined under the transmission electron microscopy （TEM）. The presence of autophagic vacuoles, as a marker of autophagy, was detected by using fluorescent dye monodansylcadaver- ine （MDC）. The variation of autophagy-related gene expression was analyzed by using real-time fluorescent quantitative polymerase chain reaction （FQ-PCR） to evaluate the effect of compression on autophagy of rab- bit NP cells. Results Compression induced rabbit NP cell degeneration and death. The autophagosomes were detected in rabbit NP cells under the TEM. The autophagic rate was （1. 580 ± 0. 171 ）% , （7. 930 ± 0. 252） %, （ 13. 530 ± 1. 206） % at 12, 24, and 48 h respectively. The mRNA expression of beclin-1 and LC3B was significantly higher in the compression treatment group than in the control group （P 〈 0. 05 ）, and the mRNA expression of beclin-1 and LC3B was increased over time under compression culture condi- tion. Conclusion Autophagy and autophagic cell death were present in compression-induced rat NP cell injury, which offers a novel sight about compression-induced degeneration of IVD cells.