重症肌无力中枢神经受损的分子机制

发布时间：2018-06-16 03:26

本文选题：重症肌无力 + 硫氧还蛋白　；参考：《昆明理工大学》2014年博士论文

【摘要】：重症肌无力(myasthenia gravis, MG)是以神经肌肉接头(neuromuscular junction, NMJ)乙酰胆碱(acetylcholine, Ach)传递障碍,导致骨骼肌无力、易疲劳等特征的自身免疫性疾病。通过临床神经心理学方法,对MG病人的记忆、注意、空间及执行功能等认知功能与患者疲劳感的相关性进行研究,表明MG存在中枢神经系统受累。胆碱能系统不仅存在于神经肌接头处,亦广泛分布于皮质、基底节、下丘脑、脑干核团、小脑等部位,并参与注意、记忆、执行、语言等认知功能。神经胶质细胞广泛分布于中枢神经系统(central nervous system, CNS),参与神经元代谢,对神经细胞具有支持、保护和营养作用。抗氧化剂和自由基之间的不平衡导致氧化应激,在神经肌肉疾病中,自由基被认为是神经肌肉结构损伤的重要原因。氧化应激也参与炎症和自身免疫介导的组织破坏。硫氧还蛋白(thioredoxin-1, Trx-1)具有多种生物学功能：调节氧化还原反应、转录因子活性、炎症反应以及抑制细胞凋亡。Trx-1还与内质网应激密切相关,抵抗内质网应激损伤。氨基末端激酶(Jun N-terminal kinase, JNK)、凋亡增强子结合蛋白同源蛋白(CCAAT/EBP homologous protein, CHOP)和含半胱氨酸的天冬氨酸蛋白水解酶-12(cysteinyl aspartate specific proteinase12, caspase-12)是内质网应激的主要分子标记。细胞凋亡与炎性反应在MG的发生和发展中起重要的作用,然而MG中枢受累的分子还不清楚。本论文研究了MG中枢受累的可能的分子机制,主要的研究结果如下： (1)选择乙酰胆碱受体抗体(AchRab)日性,且未行胸腺手术和免疫抑制剂治疗的MG患者血浆,提取AchRab,作用于神经母细胞瘤细胞SH-SY5Y,及大鼠嗜铬细胞瘤细胞PC-12,24h后细胞存活率下降。用神经生长因子(nerve growth factor, NGF)预刺激,NGF组较Ab组细胞存活率上升,提示NGF可以抵抗AChRab所致SH-SY5Y的损害。 (2)预先给予NGF,再用AChRab刺激SH-SY5Y细胞24h后,结果发现Ab组较对照组Trx-1蛋白表达增加,Ab+NGF组的Trx-1蛋白表达也增加,提示AchRab导致SH-SY5Y产生氧化应激,而NGF可以增加SH-SY5Y抵抗AChRab氧化应激损伤。 (3)预先给予NGF,再用AChRab刺激SH-SY5Y细胞24h后,结果发现：Ab组较对照组Fas、Fas、mRNA水平增加,Ab+NGF组与Ab组比较,Fas、FasL mRNA水平下降。AchRab诱导Fas/FasL增加,诱导了细胞凋亡,而NGF可以抑制AChRab所致Fas/FasL的增加,从而保护SH-SY5Y细胞免于凋亡。 (4)预先给予NGF,再用AChRab刺激大鼠神经胶质瘤细胞(C6)24h后,检测细胞因子TNF-α、IL-2,结果发现：Ab组较对照组TNF-α、IL-2mRNA水平增加,NGF组与Ab组比较,TNF-α、IL-2mRNA水平明显下降。我们的结果显示,AChRab可能增加炎症因子,而NGF抑制了fNF-a和IL-2等炎症因子的释放,从而保护了C6细胞免于AchRab的损害。 (5)应用AchR肽段构建实验性自身免疫性重症肌无力小鼠动物模型(experimental autoimmune myasthenia gravis, EAMG);并应用临床症状Lennon评分法、翻转悬挂实验、游泳实验、检测小鼠血AchRab浓度,评估EAMG模型。 (6)发现EAMG小鼠模型的脑组织额叶、海马的丙二醛(malondialdehyde,MDA)增高,Trx-1表达下降,表明在EAMG的大脑中发生了氧化应激。 (7) EAMG小鼠模型的脑组织额叶、海马的pro-caspase-3的表达减低,证实额叶、海马部位存在细胞凋亡；EAMG额叶、海马的CHOP增高,pro-caspase-12下降,说明EAMG模型小鼠中内质网途径所致细胞凋亡发生；EAMG额叶、海马pro-caspase-9亦降低,提示线粒体途径也介导了EAMG模型小鼠细胞凋亡；该结果显示：内质网和线粒体途径与EAMG额叶和海马脑组织细胞凋亡有关。 (8)发现EAMG额叶、海马磷酸化CREB(phosphorylated CREB, P-CREB)、 CREB表达下降,可能与重症肌无力记忆功能损害有关。总之,Trx-1诱导物NGF可以抑制AchRab导致的TNF-a和IL-2等炎症因子的释放,抑制细胞凋亡。EAMG脑组织额叶、海马Trx-1的表达降低,同时pro-caspase-12及pro-caspase-9的表达降低,表明Trx-1减低,以及内质网和线粒体途径与MG中枢神经系统的功能受损有关。因此,Trx=1可能是MG.新的治疗靶点。
[Abstract]:Myasthenia gravis (myasthenia gravis, MG) is an autoimmune disease characterized by the transmission of neuromuscular junction (NMJ) acetylcholine (acetylcholine, Ach), which leads to skeletal muscle weakness, fatigue and other characteristics.
Through clinical neuropsychological methods, the correlation of cognitive functions such as memory, attention, space and executive function to patients' fatigue in MG patients was studied, indicating that MG is involved in the involvement of the central nervous system.
The cholinergic system is not only located at the nerve muscle junction, but also widely distributed in cortex, basal ganglia, hypothalamus, brain stem nuclei, cerebellum, etc., and participates in cognitive functions such as attention, memory, execution, and language. Neuroglia cells are widely distributed in the central nervous system (central nervous system, CNS), involved in neuron metabolism, and on nerve cells. Support, protection and nutrition.
The imbalance between antioxidants and free radicals leads to oxidative stress. In neuromuscular diseases, free radicals are considered as an important cause of neuromuscular structural damage. Oxidative stress is also involved in inflammation and autoimmune mediated tissue damage.
Thioredoxin-1 (Trx-1) has a variety of biological functions: regulating redox reaction, transcription factor activity, inflammatory response, and inhibiting cell apoptosis,.Trx-1 is also closely related to endoplasmic reticulum stress, resistance to endoplasmic reticulum stress damage. Amino terminal kinase (JNK), apoptosis enhancer binding protein homologous eggs CCAAT/EBP homologous protein (CHOP) and cysteine - containing aspartate protein hydrolase -12 (cysteinyl aspartate specific proteinase12, caspase-12) are the main molecular markers of endoplasmic reticulum stress.
Apoptosis and inflammatory response play an important role in the occurrence and development of MG. However, the molecules involved in the MG central nervous system are not yet clear.
The possible molecular mechanism of MG central involvement is studied in this paper. The main results are as follows:
(1) AchRab was extracted from the plasma of MG patients who had selected acetylcholine receptor antibody (AchRab), without thymic surgery and immunosuppressive therapy. The survival rate of SH-SY5Y in neuroblastoma cells and the cell survival rate of pheochromocytoma cells in rats were decreased. Nerve growth factor (nerve growth factor, NGF) was used for pre stimulation, and NGF group was more Ab. The cell survival rate increased, suggesting that NGF could resist SH-SY5Y damage caused by AChRab.
(2) after giving NGF in advance and using AChRab to stimulate SH-SY5Y cell 24h, the results showed that the expression of Trx-1 protein in the Ab group increased and the Trx-1 protein expression in the Ab+NGF group increased, suggesting that AchRab led to the oxidative stress in SH-SY5Y, while NGF could increase the resistance to oxidative stress in SH-SY5Y.
(3) after giving NGF in advance and using AChRab to stimulate SH-SY5Y cell 24h, it was found that the level of Fas, Fas, mRNA in the Ab group was higher than that of the control group, and the Ab+NGF group was compared with the Ab group.
(4) given NGF in advance, and then using AChRab to stimulate the rat glioma cells (C6) 24h, the cytokine TNF- alpha and IL-2 were detected. The results showed that the level of IL-2mRNA increased in the Ab group than that in the control group, and the NGF group was compared with the Ab group, and the TNF- alpha level decreased obviously. 2 release of inflammatory factors, thereby protecting C6 cells from AchRab damage.
(5) the experimental autoimmune myasthenia gravis mice model (experimental autoimmune myasthenia gravis, EAMG) was constructed with AchR peptide segment, and the clinical symptom Lennon score was used, the reversal suspension experiment and swimming test were used to detect the concentration of AchRab in the blood of mice, and the EAMG model was evaluated.
(6) it was found that the frontal lobe of the brain tissue of the EAMG mouse model, the malondialdehyde (malondialdehyde, MDA) of the hippocampus increased and the expression of Trx-1 decreased, indicating that oxidative stress occurred in the brain of EAMG.
(7) the pro-caspase-3 expression in the frontal lobe of the brain tissue of the EAMG mouse model was reduced, and the apoptosis of the frontal lobe and hippocampus was confirmed. The CHOP in the frontal lobe, the CHOP in the hippocampus, and the pro-caspase-12 decreased, indicating the apoptosis in the endoplasmic reticulum in the EAMG model mice; the EAMG frontal lobe and the hippocampal pro-caspase-9 also decreased, suggesting mitochondria in the hippocampus, suggesting mitochondria in the hippocampus. The pathway also mediated apoptosis in EAMG mice. The results showed that endoplasmic reticulum and mitochondrial pathway were related to apoptosis in EAMG frontal and hippocampal brain tissues.
(8) EAMG CREB phosphorylated CREB (P-CREB) and CREB expression decreased in frontal lobe, which may be related to impairment of memory function in myasthenia gravis.
In conclusion, Trx-1 inducer NGF can inhibit the release of inflammatory factors such as TNF-a and IL-2 caused by AchRab, inhibit the apoptosis of the frontal lobe of.EAMG brain tissue, decrease the expression of Trx-1 in the hippocampus, and decrease the expression of pro-caspase-12 and pro-caspase-9, indicating that Trx-1 decreases, and the function of the endoplasmic reticulum and mitochondrial pathway and the MG central nervous system is impaired. So, Trx=1 may be a new therapeutic target for MG..
【学位授予单位】：昆明理工大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R746.1

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