基于iPS细胞系的亨廷顿舞蹈病发病机制研究

发布时间：2018-02-19 18:30

  本文关键词： 亨廷顿舞蹈病(HD) iPS 诱导分化 运动神经元 线粒体功能障碍　出处：《华北理工大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的基于诱导性多潜能干细胞(induced pluripotent stem cells,i PS)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 PS细胞定向诱导分化成运动神经元(motor neurons,MNS),并在诱导性多潜能干细胞和运动神经元的基础上探讨HD的发病机制。方法1以DCFH-DA、Fluo-3AM和JC-1为荧光探针,利用流式分析法分别对正常人和HD患者诱导性多潜能干细胞内活性氧(Reactive oxygen species,ROS)、Ca2+浓度和线粒体膜电位进行检测。2选用对数生长期的正常人和HD患者的i PS细胞,用EDTA将i PS细胞消化成小片状,并将其转接于低吸附的六孔板,在含DMEM/F12、Dorsomophin、N2、SB431542以及NEAA的培养基进行EB悬浮培养。在EB形成第7天补充生长因子RA。在第10天将EB贴壁培养。第13~18天补充SHH,第17天,用木瓜蛋白酶将其消化成单个细胞再贴壁培养,在神经元基础培养基上再辅以B27、BDNF和GDNF培养7天。然后用多聚甲醛固定并用免疫荧光染色法检测运动神经元特异性标记物HB9和ISL1的表达。3以DCFH-DA、Fluo-3AM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进行活性氧、Ca2+浓度和线粒体膜电位检测。结果1 HD患者诱导性多潜能干细胞内代表活性氧水平的荧光强度(1096.67±122.67)较正常组(497.33±83.50)明显增强(P(27)0.01),Ca2+浓度的荧光强度(1914.33±41.74)较正常组(978.67±38.85)明显升高(P(27)0.01),代表线粒体膜电位的红绿荧光强度比值(4.21±0.63)较正常组(7.41±0.38)相比明显降低(P(27)0.01)。2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,而且免疫荧光染色结果显示,βIII-微管蛋白(βIII-tubulin/Tuj1)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,证明亨廷顿舞蹈病和正常人特异性的i PS细胞能够诱导分化为运动神经元。3 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)明显增强(P(27)0.01),Ca2+浓度的荧光强度(2023.67±103.27)较正常组(1079.67±54.85)明显升高(P(27)0.01),代表线粒体膜电位的红绿荧光强度比值(2.74±0.13)较正常组(3.97±0.29)明显降低(P(27)0.01)。结论1 HD患者iPS细胞内出现了ROS水平和Ca2+浓度升高,及线粒体膜电位降低的线粒体功能障碍的现象。2 HD患者特异性i PS细胞能够诱导分化成为运动神经元,且为实验提供研究模型。3 HD的发病可能与运动神经元的线粒体功能障碍有关。
[Abstract]:Based on the purpose of induced pluripotent stem cells (induced pluripotent stem cells, I PS) pluripotency characteristics of Huntington disease (Huntington disease, HD) induced to differentiate into motor neurons of normal people and patients with specific I PS cells (motor neurons, MNS), and to explore the mechanism of HD based on induction pluripotent stem cells and motor neurons. Methods 1 to DCFH-DA, Fluo-3AM and JC-1 as a fluorescence probe, the use of normal human and patients with HD induced pluripotent stem cells of reactive oxygen species by flow cytometry analysis (Reactive oxygen, species, ROS), the normal concentration of mitochondrial membrane potential and Ca2+.2 were detected by using the log the growth period and in patients with HD I PS EDTA I cells, PS cells digested into small pieces, and the transfer plate in low adsorption, with DMEM/F12, Dorsomophin, N2, SB431542 and NEAA in the medium of EB suspension culture Keep seventh days. The formation of growth factor RA. in tenth days EB adherent culture in EB. The first 13~18 days of SHH, seventeenth days, with the papain digested into single cell adherent culture, neurons in the basal medium supplemented by B27, BDNF and GDNF cultured for 7 days. Then the expression of.3 poly formaldehyde fixation and immunofluorescence staining to detect motor neuron specific markers HB9 and ISL1 in DCFH-DA, Fluo-3AM and JC-1 as a fluorescence probe by flow cytometry analysis respectively on the movement of normal subjects and patients with HD neurons of active oxygen, concentration of mitochondrial membrane potential and Ca2+. The results of fluorescence intensity the potential of stem cells represent the level of reactive oxygen induced by 1 HD patients (1096.67 + 122.67) compared with the normal group (497.33 + 83.50) increased significantly (P (27) 0.01), the fluorescence intensity of the concentration of Ca2+ (1914.33 + 41.74) compared with the normal group (978.67 + 38.85) increased significantly (P (27) 0.01 ), the ratio of red to green fluorescence intensity of mitochondrial membrane potential (4.21 + 0.63) compared with the normal group (7.41 + 0.38) was significantly lower than (P (27).2 0.01) after 25 days of differentiation of motor neurons were successful in HD patients and normal people, and immunofluorescence staining showed that III- beta tubulin (beta III-tubulin/Tuj1) positive nerve cells and expression of motor neuron specific markers HB9 and ISL1, that the fluorescence intensity of Huntington disease and normal human specific I PS cells can be induced to differentiate into motor neurons in patients with motor neuron.3 HD cells represent the level of reactive oxygen species (4704.33 + 390.50) compared with the normal group (2840.33 + 166.20 (P) increased significantly (27) 0.01), the fluorescence intensity of the concentration of Ca2+ (2023.67 + 103.27) compared with the normal group (1079.67 + 54.85) increased significantly (P (27) 0.01), the ratio of red to green fluorescence intensity of mitochondrial membrane potential (2.74 + 0.13) is The normal group (3.97 + 0.29) was significantly lower (P (27) 0.01). Conclusion: 1 patients with HD in iPS cells the level of ROS and Ca2+ concentration, mitochondrial dysfunction and decreased mitochondrial membrane potential, the phenomenon of.2 in patients with HD specific I PS cells can be induced to differentiate into motor neurons, and related to mitochondrial dysfunction in experiment provide the pathogenesis of motor neurons of the.3 model of HD.

【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R742.2
，

本文编号：1517822