【作者】 周建国；

【导师】 杨操；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 硕士

【摘要】 【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。干预3天后分别采用MTT法、Annexin V-PI染色、线粒体膜电位JC-1染色和RT-PCR检测各组纤维环细胞增殖活力、细胞凋亡状况、细胞线粒体膜电位及细胞聚集蛋白多糖(aggrecan)、Ⅰ和Ⅱ型胶原基因表达。【结果】①M TT检测显示:第3、4组吸光度值(A)较正常对照组明显下降(P<0.05,P<0.05),而第5、6、7组均高于第3组(P<0.05,P<0.05,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。③荧光分光光度计分析显示:第2、3、4组红绿荧光比值较正常对照组明显下降(P<0.05,P<0.05, P<0.05),而第5、6、7组均高于第3,4组(P<0.05,P<0.05,P<0.05)。④RT-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(P<0.05,P<0.05,P<0.05),而第5、6、7组较第3组均明显增加(P<0.05,P<0.05,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NO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。更多还原

【Abstract】 Objective: To investigate the regulatory effects and mechanism of nitric oxide on the biological behaviour of rabbit annulus fibrosus(AF) cells in vitro.Methods: Rabbit AF cells gel culture model was adopted in this study. Cultured AF cells were divided into 7 groups, and various concentrations of Sodium Nitroprusside (SNP) and iNOS inhibitors (Niacinamide and 1400W) were added to the medium for intervention. After 3 days of intervention, AF cell proliferation, apoptosis, mitochondrial membrane potential and the gene expressions of aggrecan, typeⅠandⅡcollagen in each group were determined respectively by MTT assay, Annexin V-PI staining, mitochondrial membrane potential JC-1 staining and RT-PCR detection.Results:①The absorbance(A) of MTT assay in group 3 and 4 were decreased significantly as compared with the normal control group(P<0.05,P<0.05), while the A of group 5, 6 and 7 were markedly higher than that of group 3(P<0.05,P<0.05,P<0.05).②Flow cytometry analysis showed the apoptotic ratio of group 2, 3 and 4 were far higher than that of the normal control group, in which the apoptotic ratio of group 4 was 32.98%; while the apppoptotic ratio of group 5, 6 and 7 were decreased significantly as compared with group 3, but remained higher than that of the normal control group.③Fluorospectrophotometer analysis showed the red/green fluorescence ratio of group 2, 3 and 4 were obviously lower than that of the normal control group(P<0.05, P<0.05, P<0.05), while the red/green fluorescence ratio of group 5, 6 and 7 were higher than that of group 3 and 4(P<0.05,P<0.05,P<0.05).④RT-PCR showed the gene expressions of aggrecan, typeⅠa ndⅡcollagen in group 2, 3 and 4 were significantly lower than that of the normal control group(P<0.05, P<0.05, P<0.05), while the gene expressions of aggrecan, typeⅠandⅡcollagen in group 5, 6 and 7 were markedly increased as compared with group 3(P<0.05, P<0.05, P<0.05).Conclusions: NO has a strong ability of disturbing the biological behavior of rabbit AF cells, which maybe plays an important role in the AF injuried-mechanism. Inhibitors of iNOS can alleviate the disturbing effects of NO on biological behaviors of rabbit AF cells effectively, which are thought to possess the favorable potential for prevention and treatment of intervertebral disc degeneration.更多还原