摘要
目的 探讨5-甲基胞嘧啶(5-mC)水平以及DNA甲基转移酶1、3A和3B(DNMT1、DNMT3A和DNMT3B)蛋白表达变化与肌萎缩侧索硬化症(amyotrophic lateral sclerosis,ALS)之间的关系.方法 选用发病前期(70 d)、发病早期(95 d)、发病中期(108 d)和发病晚期(122 d)的hSOD1-G93A突变的ALS转基因小鼠,以同窝野生型(wild type,WT)小鼠作为对照,应用ELISA和Dot blot技术检测ALS小鼠脊髓中5-mC水平的变化规律,采用Western blot和免疫荧光双标染色技术检测ALS小鼠不同时期的脊髓中DNMT1、DNMT3A、DNMT3B的蛋白水平变化以及定位.结果 ELISA和Dot blot结果显示,与WT小鼠相比,ALS转基因小鼠脊髓中5-mC水平升高;Western blot分析显示,与WT小鼠相比,ALS转基因小鼠脊髓中DNMT1 蛋白水平在 70 d无显著性变化,在 95 d、108 d和 122 d显著升高;DNMT3A蛋白水平在70 d、95 d和108 d升高,在122 d无显著性变化;DNMT3B蛋白水平在70 d无显著性变化,在95 d、108 d和122 d显著升高.结论 ALS 转基因小鼠脊髓中5-mC水平及关键DNA甲基转移酶DNMT1、DNMT3A和DNMT3B的蛋白水平异常升高与 ALS发病密切相关.
Abstract
Objective To investigate the relationship between the levels of 5-mC and the protein expression of DNA meth-yltransferases 1,3A,and 3B(DNMT1,DNMT3A,and DNMT3B)in amyotrophic lateral sclerosis(ALS).Methods hSOD1-G93A mutation-ALS transgenic mice were selected at pre-onset(70 d),early onset(95 d),mid-onset(108 d),and late onset(122 d),with littermate WT mice as controls.ELISA and dot blot techniques were used to detect the changes in 5-mC levels in the spinal cords of the ALS mice.Western blot and immunofluorescent double staining techniques were utilized to examine the changes of protein levels and localization of DNMT1,DNMT3A,DNMT3B in the spinal cords of the ALS mice at different stages.Results ELISA and Dot blot results showed that compared to WT mice,the levels of 5-mC in the spinal cords of the ALS transgenic mice were elevated.Western blot analysis indicated that compared to WT mice,the protein levels of DNMT1 in the spinal cords of the ALS transgenic mice showed no significant change at 70 d,but were significantly increased at 95 d,108 d,and 122 d.The protein levels of DNMT3A were elevated at 70 d,95 d,and 108 d,with no significant changes at 122 d.The protein levels of DNMT3B showed no significant change at 70 d,but were significantly increased at 95 d,108 d,and 122 d.Conclusion The abnormal changes in the levels of 5-mC and the abnormally elevated protein levels of key DNA methyltransferases DNMT1,DNMT3A,and DNMT3B in the spinal cords of the ALS transgenic mice are closely associated with the onset of ALS.
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