目的:研究一氧化氮(NO)对兔纤维环细胞生物学行为的影响及其相关机制。方法:构建兔纤维环细胞凝胶培养模型并将其为7组,分别向培养基内加入不同浓度的NO供体硝普钠(SNP)及iNOS抑制剂烟酰胺和1400W进行干预:第1组为正常对照组,不加入药物;第2组加入10μmol/LSNP;第3组加入100μmol/LSNP;第4组加入200μmol/LSNP;第5组加入100μmol/LSNP和1μmol/L1400W;第6组加入100μmol/LSNP和0.05mg/mL烟酰胺;第7组加入100μmol/LSNP和0.5mg/mL烟酰胺。干预3d后分别采用MTT法、AnnexinV-PI染色和RT-PCR检测各组纤维环细胞增殖活力、细胞凋亡状况及细胞聚集蛋白多糖(aggrecan)、Ⅰ和Ⅱ型胶原基因表达。结果:①MTT检测显示:第3、4组吸光度值(A)较正常对照组明显下降(均P〈0.05),而第5、6、7组均高于第3组(均P〈0.05)。②流式细胞仪分析显示:第2、3、4组凋亡细胞比例远高于正常对照组,其中第4组高达32.98%;第5、6、7组凋亡细胞比例较第3组均有所下降,但仍高于正常对照组。③RT-PCR显示:第2、3、4组aggrecan、Ⅰ和Ⅱ型胶原基因表达较正常对照组均明显降低(均P〈0.05),而第5、6、7组较第3组均明显增加(均P〈0.05)。结论::NO对兔纤维环细胞生物学行为有较强的干扰作用,可能在纤维环损伤机制中扮演着重要角色。iNOS抑制剂可以有效抑制N0对兔纤维环细胞生物学行为的干扰,具备用于椎间盘退变防治的良好潜力。
Objective:To investigate the regulatory effects and mechanism of nitric oxide (NO) on the biological behavior of rabbit annulus fibrosus (AF) cells. Methods:Rabbit AF cells gel culture model was adopted in this study. Cultured AF cells were divided into 7 groups. Various concentrations of Sodium Nitroprusside (SNP) and iNOS inhibitors (Niacinamide and 1400W) were added to the medium as following: group 1 without any drug as the normal control group, 10μmol/L SNP in group 2, 100μmol/L SNP in group 3, 200μmol/L SNP in group 4, 100μmot/L SNP and 1μmol/L 1400W in group 5, 100μmol/L SNP and 0.05mg/mL Niacinamide in group 6, and 100μmol/L SNP and 0.5mg/mL Niacinamide in group 7. After 3 days, AF cell proliferation, apoptosis and the gene expressions of aggrecan, type Ⅰ and Ⅱ collagen in each group were determined respectively by MTT assay, Annexin V --PI staining and RT--PCR detection. Results: ①The absorbance of MTT assay in group 3 and 4 were decreased significantly compared with normal control group (P〈0.05, respectively), while the absorbance of group 5, 6 and 7 were markedly higher than that of group 3 (P〈0.05, respectively). ② Flow cytometry analysis showed the apoptotic ratio of group 2, 3 and 4 were far higher than that of normal control group, in which the apoptotic ratio of group 4 was 32.98%. And the apoptotic ratio of group 5, 6 and 7 were decreased significantly compared with group 3, but remained higher than that of normal control group. ③RT-PCR showed the gene expressions of aggrecan, typeI and Ⅱ collagen in group 2, 3 and 4 were significantly lower than that of normal control group (P〈0.05, respectively), while the gene expressions of aggrecan, typeⅠ and Ⅱ collagen in group 5, 6 and 7 were markedly increased as compared with group 3 (P〈0.05, respectively). Conclusion: NO has a strong disturbing effect on the biological behavior of rabbit AF cells, which might play an important role in the AF injury. Inhibitors of iNOS can alleviate