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目的:基于诱导性多潜能干细胞(induced pluripotent stem cells,i PSC)多潜能性的特点将亨廷顿舞蹈病(Huntington disease,HD)患者和正常人特异性i PSC定向诱导分化成运动神经元,并在运动神经元的基础上探讨HD的发病机制。方法:将HD患者和正常人的i PS细胞在特定的生长因子和神经因子的作用下定向诱导分化成运动神经元。然后用免疫荧光染色检测运动神经元特异性标记物HB9和ISL1的表达。以DCFH-DA和JC-1为荧光探针,利用流式分析法分别对正常人和HD患者运动神经元细胞活性氧和线粒体膜电位进行检测。结果:经过25天诱导分化成功得到HD患者和正常人的运动神经元,并且免疫荧光染色显示,βIII-微管蛋白阳性的神经细胞同时表达运动神经元特异性的标志物HB9和ISL1。此外,经实验统计发现HD患者运动神经元细胞内代表活性氧水平的荧光强度(4704.33±390.50)较正常组(2840.33±166.20)有明显增强(P=0.002),而且代表线粒体膜电位红绿荧光强度比(2.74±0.13)较正常组(3.97±0.29)相比有明显降低(P=0.03)。结论:HD患者特异性i PSC能够诱导分化成运动神经元,为实验提供研究模型。HD的发病与运动神经元细胞线粒体功能障碍有关。
OBJECTIVE: To induce the differentiation of motor neurons into motor neurons in patients with Huntington disease (HD) and in normal subjects based on the pluripotency of induced pluripotent stem cells (iPSC) Motor neurons based on the pathogenesis of HD. Methods: i PS cells of HD patients and normal people were induced to differentiate into motor neurons by specific growth factors and neurokines. The expression of motoneuron-specific markers HB9 and ISL1 was then detected by immunofluorescence staining. Using DCFH-DA and JC-1 as fluorescent probes, the reactive oxygen species and mitochondrial membrane potential of motor neurons in normal and HD patients were detected by flow cytometry. RESULTS: Motor neurons of HD patients and normal subjects were successfully obtained after 25 days of differentiation. Immunofluorescent staining showed that βΙΙΙ-tubulin positive neurons expressed motoneuron-specific markers HB9 and ISL1 at the same time. In addition, experimental results showed that the fluorescence intensity (4704.33 ± 390.50) in reactive oxygen species of motor neurons in HD patients was significantly higher than that in normal controls (P = 0.002) (P = 0.002), and represented the mitochondrial membrane potential of red and green fluorescence The intensity ratio (2.74 ± 0.13) was significantly lower than that of the normal group (3.97 ± 0.29) (P = 0.03). CONCLUSIONS: HD patient-specific i PSC can differentiate into motor neurons and provide a model for the experiment. The pathogenesis of HD is associated with mitochondrial dysfunction in motor neurons.