本文關(guān)鍵詞： 亨廷頓舞蹈病(HD) iPS 誘導(dǎo)分化 運(yùn)動(dòng)神經(jīng)元 線粒體功能障礙　出處：《華北理工大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：目的基于誘導(dǎo)性多潛能干細(xì)胞(induced pluripotent stem cells,i PS)多潛能性的特點(diǎn)將亨廷頓舞蹈病(Huntington disease,HD)患者和正常人特異性i PS細(xì)胞定向誘導(dǎo)分化成運(yùn)動(dòng)神經(jīng)元(motor neurons,MNS),并在誘導(dǎo)性多潛能干細(xì)胞和運(yùn)動(dòng)神經(jīng)元的基礎(chǔ)上探討HD的發(fā)病機(jī)制。方法1以DCFH-DA、Fluo-3AM和JC-1為熒光探針,利用流式分析法分別對(duì)正常人和HD患者誘導(dǎo)性多潛能干細(xì)胞內(nèi)活性氧(Reactive oxygen species,ROS)、Ca2+濃度和線粒體膜電位進(jìn)行檢測(cè)。2選用對(duì)數(shù)生長(zhǎng)期的正常人和HD患者的i PS細(xì)胞,用EDTA將i PS細(xì)胞消化成小片狀,并將其轉(zhuǎn)接于低吸附的六孔板,在含DMEM/F12、Dorsomophin、N2、SB431542以及NEAA的培養(yǎng)基進(jìn)行EB懸浮培養(yǎng)。在EB形成第7天補(bǔ)充生長(zhǎng)因子RA。在第10天將EB貼壁培養(yǎng)。第13~18天補(bǔ)充SHH,第17天,用木瓜蛋白酶將其消化成單個(gè)細(xì)胞再貼壁培養(yǎng),在神經(jīng)元基礎(chǔ)培養(yǎng)基上再輔以B27、BDNF和GDNF培養(yǎng)7天。然后用多聚甲醛固定并用免疫熒光染色法檢測(cè)運(yùn)動(dòng)神經(jīng)元特異性標(biāo)記物HB9和ISL1的表達(dá)。3以DCFH-DA、Fluo-3AM和JC-1為熒光探針,利用流式分析法分別對(duì)正常人和HD患者運(yùn)動(dòng)神經(jīng)元細(xì)胞進(jìn)行活性氧、Ca2+濃度和線粒體膜電位檢測(cè)。結(jié)果1 HD患者誘導(dǎo)性多潛能干細(xì)胞內(nèi)代表活性氧水平的熒光強(qiáng)度(1096.67±122.67)較正常組(497.33±83.50)明顯增強(qiáng)(P(27)0.01),Ca2+濃度的熒光強(qiáng)度(1914.33±41.74)較正常組(978.67±38.85)明顯升高(P(27)0.01),代表線粒體膜電位的紅綠熒光強(qiáng)度比值(4.21±0.63)較正常組(7.41±0.38)相比明顯降低(P(27)0.01)。2經(jīng)過(guò)25天誘導(dǎo)分化成功得到HD患者和正常人的運(yùn)動(dòng)神經(jīng)元,而且免疫熒光染色結(jié)果顯示,βIII-微管蛋白(βIII-tubulin/Tuj1)陽(yáng)性的神經(jīng)細(xì)胞同時(shí)表達(dá)運(yùn)動(dòng)神經(jīng)元特異性的標(biāo)志物HB9和ISL1,證明亨廷頓舞蹈病和正常人特異性的i PS細(xì)胞能夠誘導(dǎo)分化為運(yùn)動(dòng)神經(jīng)元。3 HD患者運(yùn)動(dòng)神經(jīng)元細(xì)胞內(nèi)代表活性氧水平的熒光強(qiáng)度(4704.33±390.50)較正常組(2840.33±166.20)明顯增強(qiáng)(P(27)0.01),Ca2+濃度的熒光強(qiáng)度(2023.67±103.27)較正常組(1079.67±54.85)明顯升高(P(27)0.01),代表線粒體膜電位的紅綠熒光強(qiáng)度比值(2.74±0.13)較正常組(3.97±0.29)明顯降低(P(27)0.01)。結(jié)論1 HD患者iPS細(xì)胞內(nèi)出現(xiàn)了ROS水平和Ca2+濃度升高,及線粒體膜電位降低的線粒體功能障礙的現(xiàn)象。2 HD患者特異性i PS細(xì)胞能夠誘導(dǎo)分化成為運(yùn)動(dòng)神經(jīng)元,且為實(shí)驗(yàn)提供研究模型。3 HD的發(fā)病可能與運(yùn)動(dòng)神經(jīng)元的線粒體功能障礙有關(guān)。
[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.

【學(xué)位授予單位】：華北理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R742.2
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本文編號(hào)：1517822