本文選題：帕金森病 + 小膠質(zhì)細胞　； 參考：《青島大學》2017年碩士論文

【摘要】：帕金森病(parkinson’s disease,PD)是一種多發(fā)于中老年的中樞神經(jīng)系統(tǒng)退行性疾病,以運動不能、肌僵直、靜止性震顫及姿勢反射障礙為特征性表現(xiàn),其病理學特征是中腦黑質(zhì)致密帶(substantia nigra pars compacta,SNpc)多巴胺(dopamine,DA)能神經(jīng)元脫失。迄今為止,雖然遺傳、環(huán)境和氧化應激等因素對PD發(fā)病均起到一定的作用,但其確切的病因尚不完全清楚。腦內(nèi)高鐵導致黑質(zhì)-紋狀體系統(tǒng)DA能神經(jīng)元功能的損傷已成為越來越受到神經(jīng)科學家關(guān)注的熱點問題。鐵,作為人體所必需的營養(yǎng)元素之一,廣泛地參與人體內(nèi)的生理功能和生化反應。研究發(fā)現(xiàn)鐵的異常聚積與PD的發(fā)病密切相關(guān)。鐵選擇性聚積可能與二價金屬轉(zhuǎn)運蛋白1(divalent metal transporter,DMT1)和鐵轉(zhuǎn)出蛋白ferroportin 1(FPN1)的異常表達有關(guān)。鐵調(diào)節(jié)蛋白(iron regulatory proteins,IRPs)能特異性的識別鐵轉(zhuǎn)運相關(guān)蛋白m RNA上的IRE序列,當IRPs激活,與FPN1 m RNA的5'UTR的IRE結(jié)合,使FPN1m RNA不能翻譯,FPN1蛋白表達降低;當與DMT1 m RNA的3'UTR結(jié)合時,增加其m RNA的穩(wěn)定性,DMT1蛋白表達增加。鐵調(diào)素(hepcidin)是近年來發(fā)現(xiàn)的鐵調(diào)節(jié)激素,可以抑制FPN1基因的轉(zhuǎn)錄與翻譯過程,促進FPN1的內(nèi)化和降解,降低FPN1,在維持機體鐵穩(wěn)態(tài)的平衡中發(fā)揮重要的作用。目前PD鐵沉積的研究主要集中在DA能神經(jīng)元,實際上腦內(nèi)多種膠質(zhì)細胞在鐵穩(wěn)態(tài)調(diào)節(jié)中也發(fā)揮重要作用。PD患者的中腦SNpc有大量的激活的小膠質(zhì)細胞,這些激活的小膠質(zhì)細胞有大量的鐵沉積,然而小膠質(zhì)細胞激活和鐵聚積之間的關(guān)系尚未闡明。本室前期研究表明,6羥基多巴胺(6-hydroxydopamine,6-OHDA)激活鐵調(diào)節(jié)蛋白1(iron responsive protein1,IRP1)使DMT1的表達增加,FPN1的表達降低,導致了DA能神經(jīng)元鐵的聚集及神經(jīng)元損傷。6-OHDA作用下,星形膠質(zhì)細胞DMT1和FPN1的表達均增加,鐵轉(zhuǎn)運能力增強。那么,小膠質(zhì)細胞在腦鐵代謝以及PD黑質(zhì)鐵聚積過程中是如何發(fā)揮作用的?本研究應用經(jīng)典的神經(jīng)毒性藥物6-OHDA處理小鼠小膠質(zhì)細胞系BV2細胞,采用細胞培養(yǎng)、酶聯(lián)免疫吸附實驗、實時熒光定量PCR、Western blot等多項研究方法,探究6-OHDA對BV2小膠質(zhì)細胞鐵代謝的影響。結(jié)果如下:1.10μmol/L 6-OHDA處理BV2小膠質(zhì)細胞24 h,DMT1的蛋白表達明顯上調(diào),與對照組相比,差異有統(tǒng)計學意義(P0.01)。FPN1蛋白表達水平與正常對照組相比無明顯變化(P0.05)。2.10μmol/L 6-OHDA處理BV2小膠質(zhì)細胞24 h,IRP1的蛋白表達明顯上調(diào),與對照組相比,差異有統(tǒng)計學意義(P0.01)。3.10μmol/L 6-OHDA處理BV2小膠質(zhì)細胞24h,增加細胞對鐵的攝取能力,與對照組相比,差別有統(tǒng)計學意義(P0.01),6-OHDA處理BV2小膠質(zhì)細胞,細胞的鐵轉(zhuǎn)出能力無明顯變化。(P0.05)。4.10μmol/L 6-OHDA處理BV2小膠質(zhì)細胞24h,與對照組相比,hepcidin的釋放量明顯下降,差異有統(tǒng)計學意義(P0.01)。5.10μmol/L的6-OHDA處理BV2小膠質(zhì)細胞24h,TNF-αm RNA的水平顯著升高,與正常對照組相比,差異有統(tǒng)計學意義(P0.01)。IL-1βm RNA的水平顯著升高,與正常對照組相比,差別有統(tǒng)計學意義(P0.01)。6.100μmol/L FAC處理BV2小膠質(zhì)細胞24h,DMT1蛋白表達明顯降低,與對照組相比,差異有統(tǒng)計學意義(P0.01)。FPN1蛋白表達明顯升高,與對照組相比,差異有統(tǒng)計學意義(P0.01)。7.100μmol/L FAC處理BV2小膠質(zhì)細胞24h,IRP1的蛋白表達明顯降低,與正常對照組相比,差異有統(tǒng)計學意義(P0.001)。8.100μmol/L DFO處理BV2小膠質(zhì)細胞24h,DMT1蛋白表達明顯升高,與對照組相比,差異有統(tǒng)計學意義(P0.01)。FPN1蛋白表達明顯降低,與對照組相比,差異有統(tǒng)計學意義(P0.01)。9.100μmol/L DFO處理BV2小膠質(zhì)細胞24h,IRP1的蛋白表達明顯升高,與正常對照組相比,差異有統(tǒng)計學意義(P0.01)。上述結(jié)果表明,6-OHDA處理BV2小膠質(zhì)細胞DMT1表達增加,鐵轉(zhuǎn)入增加,可能與IRP1上調(diào)有關(guān)。6-OHDA對FPN1表達無明顯作用,鐵轉(zhuǎn)出不變,可能是IRP1上調(diào),hepcidin下調(diào)共同作用的結(jié)果。6-OHDA能激活BV2小膠質(zhì)細胞,使細胞TNF-α,IL-1β表達增加。高鐵抑制了IRP1,使DMT1蛋白表達降低,FPN1的蛋白表達升高。低鐵激活了IRP1,使DMT1蛋白表達升高,FPN1的蛋白表達降低。綜上所述,6-OHDA通過激活I(lǐng)RP1,抑制hepcidin,調(diào)節(jié)了DMT1和FPN1的表達。引起小膠質(zhì)細胞鐵聚積。6-OHDA能激活小膠質(zhì)細胞,釋放炎性因子,進而造成DA神經(jīng)元的損傷。
[Abstract]:Parkinson's disease (Parkinson 's disease, PD) is a degenerative disease of the central nervous system that frequently occurs in the middle and old age. It is characterized by motor inability, muscle stiffness, static tremor and postural reflex, and its pathological features are the removal of neurons from the mesencephalic dense zone of the substantia nigra (substantia nigra pars compacta, SNpc) dopamine (dopamine, DA). So far, although heredity, environment and oxidative stress have played a certain role in the pathogenesis of PD, the exact cause is not completely clear. The damage of DA energy in the substantia nigrostriatal system has become a hot issue in neuroscientists. Iron, as a necessary battalion for the human body. One of the nutrient elements, widely involved in the physiological and biochemical reactions within the human body. Studies have found that the abnormal accumulation of iron is closely related to the incidence of PD. Selective accumulation of iron may be related to the abnormal expression of two valence metal transporter 1 (divalent metal transporter, DMT1) and iron transfer protein ferroportin 1 (FPN1). Iron regulatory protein (iron regula) Tory proteins, IRPs) can specifically identify the IRE sequence on the iron transport related protein M RNA. When IRPs is activated, it can not be translated from FPN1 m RNA 5'UTR IRE, and the expression of protein is reduced. Iron regulating hormones can inhibit the transcription and translation of the FPN1 gene, promote the internalization and degradation of FPN1, reduce FPN1, and play an important role in maintaining the balance of iron homeostasis in the body. At present, the study of PD iron deposition is mainly concentrated on the DA energy neurons. In fact, many kinds of gelatin cells in the brain also play an important role in the regulation of iron homeostasis,.PD The SNpc of the midbrain of the patient has a large number of activated microglia, and the activated microglia has a large amount of iron deposition. However, the relationship between the activation of microglia and the accumulation of iron has not been clarified. The previous study in this room showed that 6 hydroxyl dopamine (6-hydroxydopamine, 6-OHDA) activated the iron regulating protein 1 (iron responsive protein1, IRP1) to make D The expression of MT1 increased and the expression of FPN1 decreased, which led to the aggregation of iron in the DA neurons and the effect of neuron damage on.6-OHDA. The expression of DMT1 and FPN1 in astrocytes increased and the iron transport capacity increased. Then, how do microglia play a role in the process of iron metabolism and the accumulation of PD black matter iron? This study applied the classical study. Neurotoxic drug 6-OHDA treated mouse microglia BV2 cells, using cell culture, enzyme-linked immunosorbent assay, real-time fluorescence quantitative PCR, Western blot and many other research methods to explore the effect of 6-OHDA on the iron metabolism of BV2 microglia. The results were as follows: 1.10 micron 6-OHDA treated BV2 microglia 24 h, DMT1 protein expression Compared with the control group, the difference was statistically significant (P0.01), the expression level of.FPN1 protein was not significantly changed compared with the normal control group (P0.05).2.10 mu mol/L 6-OHDA treated BV2 microglia 24 h, and the expression of IRP1 protein was obviously up, and the difference was statistically significant compared with the control group (P0.01).3.10 micronux treatment Cell 24h, increasing the ability of cell uptake of iron, compared with the control group, the difference was statistically significant (P0.01), 6-OHDA treatment of BV2 microglia, the iron transfer ability of cells had no significant changes. (P0.05).4.10 mu mol/L 6-OHDA treated BV2 microglia 24h, compared with the control group, hepcidin release decreased significantly (P0.01), the difference was statistically significant (P0.01) Compared with the normal control group, the level of 24h and TNF- alpha m RNA in the 6-OHDA treated BV2 microglia was significantly higher than that in the normal control group (P0.01), and the level of.IL-1 beta m RNA was significantly higher than that of the normal control group. Compared with the normal control group, the difference was statistically significant. Lower, compared with the control group, the difference was statistically significant (P0.01).FPN1 protein expression significantly increased, compared with the control group, the difference was statistically significant (P0.01).7.100 mu mol/L FAC processing BV2 microglia 24h, the IRP1 protein expression significantly decreased, compared with the normal control group, the difference was statistically significant (P0.001).8.100 micron mol/L The expression of 24h and DMT1 protein in glial cells increased significantly. Compared with the control group, the difference was statistically significant (P0.01), the expression of.FPN1 protein decreased significantly. Compared with the control group, the difference was statistically significant (P0.01).9.100 mu mol/L DFO treated BV2 microglia 24h, IRP1 protein expression was significantly increased, compared with the normal control group, the difference was statistically significant. P0.01. The results showed that the expression of DMT1 in BV2 microglia was increased by 6-OHDA treatment, and the transfer of iron was increased. It was possible that the expression of.6-OHDA had no obvious effect on FPN1 expression with the up regulation of IRP1. It might be the up-regulation of IRP1 and the common effect of hepcidin downregulation, and.6-OHDA could stimulate the active BV2 microglia and increase the cell TNF- alpha and the expression of beta. Iron inhibits IRP1, reduces the expression of DMT1 protein, increases the expression of FPN1 protein. Low iron activates IRP1, increases the expression of DMT1 protein, and reduces the expression of FPN1 protein. In summary, 6-OHDA regulates DMT1 and FPN1 by activating IRP1, inhibiting hepcidin, and causing microglia to activate microglia and release inflammatory causes. In addition, it causes damage to DA neurons.

【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R742.5

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