DDX3和酪蛋白激酶1ε在肌萎缩侧索硬化症转基因鼠脑干中的表达变化

Expression of DDX3 and casein kinase 1ε in the brain stem of amyotrophic lateral sclerosis transgenic mice

目的检测DDX3和酪蛋白激酶1ε(CK1ε)在肌萎缩侧索硬化症(ALS)转基因鼠脑干中的表达变化,探讨DDX3和CK1ε在ALS脑干运动神经元变性中的作用。方法选取ALS转基因鼠33只,分别于发病早期(95 d)、中期(108 d)和晚期(122 d)3个时间点剥离脑干,应用RT-PCR、Western blotting和免疫荧光染色技术分别检测ALS转基因鼠脑干组织中DDX3和CK1ε的表达规律,在脑干运动核团舌下神经(12 N)和面神经(7 N)中阳性细胞的分布特点及细胞定位,每组均选择相同数量的同窝野生型鼠作为对照。结果 RT-PCR和Western blotting结果显示,与同窝野生型鼠相比,ALS转基因鼠脑干组织中DDX3和CK1εmRNA于95 d、108 d、122 d表达均无明显变化,DDX3和CK1ε蛋白在95 d和108 d表达上调,122 d表达下调(P<0.01,P<0.001)。免疫荧光结果显示,在ALS鼠和野生型鼠脑干的12 N和7N区域均可检测到DDX3和CK1ε阳性细胞,DDX3和CK1ε表达在神经元,在星形胶质细胞不表达。ALS鼠和野生型鼠DDX3和CK1ε免疫反应性具有差异。结论 DDX3和CK1ε在脑干中的表达异常与ALS发病密切相关。

Objective To detect the expression of DDX3 and casein kinase 1ε（ CK1ε） in the brain stem of amyotrophic lateral sclerosis（ ALS） transgenic mice and study the role of DDX3 and CK1ε in the degeneration of brain stem motor neurons of amyotrophic lateral sclerosis（ ALS）. Methods Thirty-three ALS transgenic mice were used in this study. The brain stem was dissected and collected at the early（ 95 days）,middle（ 108 days） or late（ 122 days） stages.The expression of DDX3 and CK1ε in the brain stem of ALS transgenic mice,the distribution and co-localization of positive cells in the hypoglossal nucleus and facial nucleus of the brain stem were detected by RT-PCR,Western blotting and immunofluorescence technology. In each group,the same number of wild type littermates were selected as controls. Results The result of RT-PCR and Western blotting showed that compared with the wild type mice,the expression of DDX3 and CK1ε mRNA in the brain stem of ALS mice remained unchanged at day 95,day 108 and day 122. DDX3 and CK1ε protein levels were up-regulated at day 95 and day 108 but down-regulated at day 122 in the ALS mice brain stem group（ P 〈0. 01,P 〈 0. 001）. The result of immunofluorescence showed that DDX3 and CK1ε positive cells were detected in the sublingual nerve and facial nerve of ALS mice and wild type mice brain stem. DDX3 and CK1ε were expressed in neurons,but not in astrocytes. The immunoreactivity of both DDX3 and CK1ε of ALS mice and wild type mice was different.Conclusion The abnormal expression of DDX3 and CK1ε in the brain stem is closely related to the pathogenesis of ALS.