本文選題：重癥肌無力 + 硫氧還蛋白　； 參考：《昆明理工大學(xué)》2014年博士論文

【摘要】：重癥肌無力(myasthenia gravis, MG)是以神經(jīng)肌肉接頭(neuromuscular junction, NMJ)乙酰膽堿(acetylcholine, Ach)傳遞障礙,導(dǎo)致骨骼肌無力、易疲勞等特征的自身免疫性疾病。 通過臨床神經(jīng)心理學(xué)方法,對MG病人的記憶、注意、空間及執(zhí)行功能等認(rèn)知功能與患者疲勞感的相關(guān)性進(jìn)行研究,表明MG存在中樞神經(jīng)系統(tǒng)受累。 膽堿能系統(tǒng)不僅存在于神經(jīng)肌接頭處,亦廣泛分布于皮質(zhì)、基底節(jié)、下丘腦、腦干核團(tuán)、小腦等部位,并參與注意、記憶、執(zhí)行、語言等認(rèn)知功能。神經(jīng)膠質(zhì)細(xì)胞廣泛分布于中樞神經(jīng)系統(tǒng)(central nervous system, CNS),參與神經(jīng)元代謝,對神經(jīng)細(xì)胞具有支持、保護(hù)和營養(yǎng)作用。 抗氧化劑和自由基之間的不平衡導(dǎo)致氧化應(yīng)激,在神經(jīng)肌肉疾病中,自由基被認(rèn)為是神經(jīng)肌肉結(jié)構(gòu)損傷的重要原因。氧化應(yīng)激也參與炎癥和自身免疫介導(dǎo)的組織破壞。 硫氧還蛋白(thioredoxin-1, Trx-1)具有多種生物學(xué)功能：調(diào)節(jié)氧化還原反應(yīng)、轉(zhuǎn)錄因子活性、炎癥反應(yīng)以及抑制細(xì)胞凋亡。Trx-1還與內(nèi)質(zhì)網(wǎng)應(yīng)激密切相關(guān),抵抗內(nèi)質(zhì)網(wǎng)應(yīng)激損傷。氨基末端激酶(Jun N-terminal kinase, JNK)、凋亡增強(qiáng)子結(jié)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)是內(nèi)質(zhì)網(wǎng)應(yīng)激的主要分子標(biāo)記。 細(xì)胞凋亡與炎性反應(yīng)在MG的發(fā)生和發(fā)展中起重要的作用,然而MG中樞受累的分子還不清楚。 本論文研究了MG中樞受累的可能的分子機(jī)制,主要的研究結(jié)果如下： (1)選擇乙酰膽堿受體抗體(AchRab)日性,且未行胸腺手術(shù)和免疫抑制劑治療的MG患者血漿,提取AchRab,作用于神經(jīng)母細(xì)胞瘤細(xì)胞SH-SY5Y,及大鼠嗜鉻細(xì)胞瘤細(xì)胞PC-12,24h后細(xì)胞存活率下降。用神經(jīng)生長因子(nerve growth factor, NGF)預(yù)刺激,NGF組較Ab組細(xì)胞存活率上升,提示NGF可以抵抗AChRab所致SH-SY5Y的損害。 (2)預(yù)先給予NGF,再用AChRab刺激SH-SY5Y細(xì)胞24h后,結(jié)果發(fā)現(xiàn)Ab組較對照組Trx-1蛋白表達(dá)增加,Ab+NGF組的Trx-1蛋白表達(dá)也增加,提示AchRab導(dǎo)致SH-SY5Y產(chǎn)生氧化應(yīng)激,而NGF可以增加SH-SY5Y抵抗AChRab氧化應(yīng)激損傷。 (3)預(yù)先給予NGF,再用AChRab刺激SH-SY5Y細(xì)胞24h后,結(jié)果發(fā)現(xiàn)：Ab組較對照組Fas、Fas、mRNA水平增加,Ab+NGF組與Ab組比較,Fas、FasL mRNA水平下降。AchRab誘導(dǎo)Fas/FasL增加,誘導(dǎo)了細(xì)胞凋亡,而NGF可以抑制AChRab所致Fas/FasL的增加,從而保護(hù)SH-SY5Y細(xì)胞免于凋亡。 (4)預(yù)先給予NGF,再用AChRab刺激大鼠神經(jīng)膠質(zhì)瘤細(xì)胞(C6)24h后,檢測細(xì)胞因子TNF-α、IL-2,結(jié)果發(fā)現(xiàn)：Ab組較對照組TNF-α、IL-2mRNA水平增加,NGF組與Ab組比較,TNF-α、IL-2mRNA水平明顯下降。我們的結(jié)果顯示,AChRab可能增加炎癥因子,而NGF抑制了fNF-a和IL-2等炎癥因子的釋放,從而保護(hù)了C6細(xì)胞免于AchRab的損害。 (5)應(yīng)用AchR肽段構(gòu)建實(shí)驗(yàn)性自身免疫性重癥肌無力小鼠動物模型(experimental autoimmune myasthenia gravis, EAMG);并應(yīng)用臨床癥狀Lennon評分法、翻轉(zhuǎn)懸掛實(shí)驗(yàn)、游泳實(shí)驗(yàn)、檢測小鼠血AchRab濃度,評估EAMG模型。 (6)發(fā)現(xiàn)EAMG小鼠模型的腦組織額葉、海馬的丙二醛(malondialdehyde,MDA)增高,Trx-1表達(dá)下降,表明在EAMG的大腦中發(fā)生了氧化應(yīng)激。 (7) EAMG小鼠模型的腦組織額葉、海馬的pro-caspase-3的表達(dá)減低,證實(shí)額葉、海馬部位存在細(xì)胞凋亡；EAMG額葉、海馬的CHOP增高,pro-caspase-12下降,說明EAMG模型小鼠中內(nèi)質(zhì)網(wǎng)途徑所致細(xì)胞凋亡發(fā)生；EAMG額葉、海馬pro-caspase-9亦降低,提示線粒體途徑也介導(dǎo)了EAMG模型小鼠細(xì)胞凋亡；該結(jié)果顯示：內(nèi)質(zhì)網(wǎng)和線粒體途徑與EAMG額葉和海馬腦組織細(xì)胞凋亡有關(guān)。 (8)發(fā)現(xiàn)EAMG額葉、海馬磷酸化CREB(phosphorylated CREB, P-CREB)、 CREB表達(dá)下降,可能與重癥肌無力記憶功能損害有關(guān)。 總之,Trx-1誘導(dǎo)物NGF可以抑制AchRab導(dǎo)致的TNF-a和IL-2等炎癥因子的釋放,抑制細(xì)胞凋亡。EAMG腦組織額葉、海馬Trx-1的表達(dá)降低,同時(shí)pro-caspase-12及pro-caspase-9的表達(dá)降低,表明Trx-1減低,以及內(nèi)質(zhì)網(wǎng)和線粒體途徑與MG中樞神經(jīng)系統(tǒng)的功能受損有關(guān)。因此,Trx=1可能是MG.新的治療靶點(diǎn)。
[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..
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R746.1

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