【作者】 王毅鹏；

【导师】 彭丹；

【作者基本信息】 中南大学 ， 骨科， 2008， 硕士

【摘要】 目的:通过用DNA甲基化抑制剂5-氮杂脱氧胞苷诱导乳兔软骨细胞凋亡,探讨DNA低甲基化在骨关节炎发病机制中的作用。方法:第一代乳兔软骨细胞随机分为对照组,5-氮杂脱氧胞苷处理组,分别培养3天。采用MTT法检测细胞存活率,电镜观察5-氮杂脱氧胞苷致软骨细胞凋亡的作用,硝酸还原酶法(NO试剂盒)测定5-氮杂脱氧胞苷作用于软骨细胞培养基上清液中NO含量,免疫印记法(Western Blotting)检测5-氮杂脱氧胞苷对软骨细胞表达一氧化氮合酶(iNOS)和Fas蛋白的影响。SPSS软件统计分析5-氮杂脱氧胞苷诱导软骨细胞凋亡与其分泌NO、iNOS和Fas蛋白的相关性。结果:1.随5-氮杂脱氧胞苷浓度增加和作用时间延长,软骨细胞存活率下降,并呈浓度及时间依赖关系(p＜0.05)。2.5-氮杂脱氧胞苷可引起软骨细胞发生凋亡的典型电镜形态改变。3.5-氮杂脱氧胞苷可刺激软骨细胞分泌NO(p＜0.05)。4.5-氮杂脱氧胞苷可使凋亡相关蛋白iNOS蛋白表达增多(p＜0.05),但不能使Fas蛋白表达增多(p＞0.10)。5.5-氮杂脱氧胞苷诱导软骨细胞的凋亡与软骨细胞分泌NO和表达iNOS蛋白正相关性(p＜0.05);与Fas蛋白的表达没有明显相关性(p＞0.10)。结论:DNA低甲基化可通过NO途径诱导兔软骨细胞凋亡。更多还原

【Abstract】 Objectives To investigate the role of hypomethylation in DNA methylation inhibitor 5-aza-CdR inducing apoptosis in cartilage cells of rabbit in OA.Methods Rabbit cartilage cells were treated with different concentrations of 5-aza-CdR at different time points(1-3d).Cell viability was measured by MTT assay.Apoptosis changes of the treated cells were observed by electron microscropy.The levels of NO in culture media were detected by nitrate reductase method.Nitric oxide synthase(iNOS)and Fas protein were measured by Western blotting assay.The relationship among 5-aza-CdR and NO and Fas was investigated by SPSS statistics assay.Results1.The results shows the dose-dependent and time-dependent effects in cell viability of 5-aza-CdR treated cartilage cells(p＜0.05).2.Apoptosis body was found in 5-aza-CdR-treated cells by electron microscropy.3.The level of NO in culture medium of 5-aza-CdR treated cells were higher than that of normal control.(p＜0.05)4.Overexpressions of iNOS protein were detected in 5-aza-CdR treated cells(p＜0.05),Overexpressions of Fas protein cannot be detected in 5aza-CdR treated cells(p＞0.10). 5.5-aza-CdR induced apoptosis was correlated with the level of NO production and the level of iNOS protein expression(p＜0.05),and had no correlation with Fas protein(p＞0.10).ConclusionsDNA hypomethylation participate in inducing cartilage cells apoptosis by enhancing NO production and the expression of iNOS.更多还原