摘要
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： The cells from Huntington disease（HD） patient and healthy people were induced to differentiate into motor neurons based on pluripotency characteristic of i PSC, and the motor neurons was used to explore the pathogenesis of HD. Methods： The i PS cells collected from HD patient and healthy people were differentiated into motor neurons under the action of certain growth factors and neurotrophic factors. The expression levels of motor neuron specific markers HB9 and ISL1 were evaluated by immunofluorescence staining. Reactive oxygen species and mitochondrial membrane potential of control and HD patient motor neurons were evaluated by flow cytometry, and DCFH-DA and JC-1 were used as the fluorescent probes respectively. Results： Successfully differentiated HD patient and control specific i PS cells into motor neurons after 25 days. And immunofluorescence staining showed that βIII-tubulin positive neurons express both motor neuron specific markers HB9 and ISL1. Furthermore, the ROS levels fluorescence intensity of HD patient motor neurons（4704.33±390.50） had significantly enhanced. than controls（2840.33±166.20）（P=0.002）, on behalf of the mitochondrial membrane potential of fluorescence intensity red-green ratio（2.74±0.13） had significantly reduced than controls（3.97±0.29）（P=0.03）. Conclusions： HD patient-specific i PSC can be differentiated into motor neurons, and provided a model for experimental research. The pathogenesis of Huntington＇s disease was associated with mitochondrial dysfunction of motor neurons.
出处
《现代生物医学进展》
CAS
2016年第33期6418-6423,共6页
Progress in Modern Biomedicine
基金
国家自然科学基金项目(81473042)
国家自然科学基金项目(31171048)
河北省自然科学基金项目(H2013209040)
