烟酰胺对兔髓核细胞增殖及凋亡的调节作用

背景：研究表明，烟酰胺能够对白细胞介素1B或肿瘤坏死因子α致椎间盘退变起到保护作用，但烟酰胺对椎间盘细胞凋亡与增殖的保护机制尚不很清楚。目的：观察烟酰胺对兔髓核细胞增殖及凋亡的调控作用及其机制。设计、时间及地点：随机对照分组设计，于2007—04／10在华中科技大学同济医学院附属协和医院骨科实验室及协和医院干细胞中心完成。材料：2—3月龄日本大白兔10只，体质量1．5-2．0kg，取L1-L6节段椎间盘内髓核细胞，体外培养髓核细胞凋亡模型。方法：实验分为6组，正常对照组：不加任何药物：烟酰胺组：加入0．5g／L烟酰胺；白细胞介素1β组：加入10μg／L白细胞介素1β：白细胞介素1β＋Caspase抑制剂组：加入10μg/L自细胞介素1β及非特异性Caspase抑制剂Z-VAD-FM；白细胞介素1β＋小剂量烟酰胺组：加入10μg/L白细胞介素1β及0．05g／L烟酰胺组：白细胞介素1β＋大剂量烟酰胺组：10μg／L白细胞介素1β及0．5g／L烟酰胺。3d后对各组细胞行Annexin V-PI染色，Caspase-3、Caspase-8、Caspase-9功能染色检测及MTT检测。主要观察指标：各组细胞凋亡率、caspase-3、8、9功能染色阳性细胞率及各组细胞的吸光度。结果：①白细胞介素1β＋Caspase抑制剂组和白细胞介素lB＋大剂量烟酰胺组较白细胞介素1β组细胞凋亡率明显下降（P〈0．01）。②白细胞介素1β＋Caspase抑制剂组、白细胞介素1β＋大、小剂量烟酰胺组Caspase-3、Caspase-8、Caspase-9功能染色阳性细胞率较白细胞介素1β组明显下降（P〈0．01或P〈0．05）。③与白细胞介素1β组相比，白细胞介素1β＋Caspase抑制剂组和白细胞介素1β＋大剂量烟酰胺组吸光度升高（P〈0．01）。结论：烟酰胺可以促进髓核细胞的增殖并抑制白细胞介素1β诱导的髓核细胞凋亡，对凋亡的抑制作用主要通过抑制凋亡的线粒体途径实现。

Regulatory effect of nicotinamide on apoptosis and proliferation of rabbit nucleus pulposus cell in vitro

BACKGROUND: It has reported that nicotinamide is capable of protecting intervertebral disc (IVD) against interleukin-1β (IL-1β) or tumor necrosis factor-alpha (TNF-α) induced degeneration. However, the protective mechanism of nicotinamide on IVD cells apoptosis and proliferation remains unclear.OBJECTIVE: To investigate regulatory effects of nicotinamide on rabbit nucleus pulposus cell apoptosis and proliferation in vitro.DESIGN, TIME AND SETTING: Randomized control grouping design, which was carried out in the Laboratory of Orthopaedics and Stem Cell Center, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology from April to October 2007.MATERIALS: Ten Japanese white rabbits (aged 2-3 months weighing 1.5-2.0 kg) were used in this study. Furthermore, nucleus pulposus cells obtained from L1-6 lumbar spine were harvested and cultured for further experiments.METHODS: The NP cells were divided into 6 groups, including control group (without any drug as control), nicotinamide group (0.5 g/L nicotinamide), IL-1β group (10 μg/L IL-1β), IL-1β + caspase group (10 μg/L IL-1β and non-specific caspase inhibitor Z-VAD-FMK), IL-1β + small-dose nicotinamide group (10 μg/L IL-1β and 0.05 g/L nicotinamide), and IL-1β + large-dose nicotinamide group (10 μg/L IL-1β and 0.5 g/L nicotinamide). After 3 days of culture, the cells were examined with Annexin V-PI staining, caspase-3, 8 and 9 activity staining and MTT assay.MAIN OUTCOME MEASURES: The apoptotic rates, the positive rates of caspase-3, 8 and 9 activity staining and the absorbance of MTT assay of each group.RESULTS: ① As compared to IL-1β group, the apoptotic rates were decreased in the IL-1β + caspase group and IL-1β + large-dose nicotinamide group (P < 0.01). ②As compared to IL-1β group, the positive rates of caspase-3, 8 and 9 activity staining were decreased in the IL-1β + caspase group, IL-1β + large-dose and small-dose nicotinamide groups (P < 0.01 or P < 0.01). ③As compared to IL-1β group, the absorbance was increased in the IL-1β + caspase group and IL-1β + large-dose nicotinamide group (P < 0.01).CONCLUSION: Nicotinamide is capable of promoting cell proliferation and inhibiting IL-1β induced apoptosis of nucleus pulposus cells in vitro. The inhibition of apoptosis mainly acts via inhibition of the mitochondrial pathway.