6-OHDA对BV2小胶质细胞铁代谢的影响

发布时间：2018-05-12 06:17

本文选题：帕金森病 + 小胶质细胞　；参考：《青岛大学》2017年硕士论文

【摘要】：帕金森病(parkinson’s disease,PD)是一种多发于中老年的中枢神经系统退行性疾病,以运动不能、肌僵直、静止性震颤及姿势反射障碍为特征性表现,其病理学特征是中脑黑质致密带(substantia nigra pars compacta,SNpc)多巴胺(dopamine,DA)能神经元脱失。迄今为止,虽然遗传、环境和氧化应激等因素对PD发病均起到一定的作用,但其确切的病因尚不完全清楚。脑内高铁导致黑质-纹状体系统DA能神经元功能的损伤已成为越来越受到神经科学家关注的热点问题。铁,作为人体所必需的营养元素之一,广泛地参与人体内的生理功能和生化反应。研究发现铁的异常聚积与PD的发病密切相关。铁选择性聚积可能与二价金属转运蛋白1(divalent metal transporter,DMT1)和铁转出蛋白ferroportin 1(FPN1)的异常表达有关。铁调节蛋白(iron regulatory proteins,IRPs)能特异性的识别铁转运相关蛋白m RNA上的IRE序列,当IRPs激活,与FPN1 m RNA的5'UTR的IRE结合,使FPN1m RNA不能翻译,FPN1蛋白表达降低;当与DMT1 m RNA的3'UTR结合时,增加其m RNA的稳定性,DMT1蛋白表达增加。铁调素(hepcidin)是近年来发现的铁调节激素,可以抑制FPN1基因的转录与翻译过程,促进FPN1的内化和降解,降低FPN1,在维持机体铁稳态的平衡中发挥重要的作用。目前PD铁沉积的研究主要集中在DA能神经元,实际上脑内多种胶质细胞在铁稳态调节中也发挥重要作用。PD患者的中脑SNpc有大量的激活的小胶质细胞,这些激活的小胶质细胞有大量的铁沉积,然而小胶质细胞激活和铁聚积之间的关系尚未阐明。本室前期研究表明,6羟基多巴胺(6-hydroxydopamine,6-OHDA)激活铁调节蛋白1(iron responsive protein1,IRP1)使DMT1的表达增加,FPN1的表达降低,导致了DA能神经元铁的聚集及神经元损伤。6-OHDA作用下,星形胶质细胞DMT1和FPN1的表达均增加,铁转运能力增强。那么,小胶质细胞在脑铁代谢以及PD黑质铁聚积过程中是如何发挥作用的?本研究应用经典的神经毒性药物6-OHDA处理小鼠小胶质细胞系BV2细胞,采用细胞培养、酶联免疫吸附实验、实时荧光定量PCR、Western blot等多项研究方法,探究6-OHDA对BV2小胶质细胞铁代谢的影响。结果如下:1.10μmol/L 6-OHDA处理BV2小胶质细胞24 h,DMT1的蛋白表达明显上调,与对照组相比,差异有统计学意义(P0.01)。FPN1蛋白表达水平与正常对照组相比无明显变化(P0.05)。2.10μmol/L 6-OHDA处理BV2小胶质细胞24 h,IRP1的蛋白表达明显上调,与对照组相比,差异有统计学意义(P0.01)。3.10μmol/L 6-OHDA处理BV2小胶质细胞24h,增加细胞对铁的摄取能力,与对照组相比,差别有统计学意义(P0.01),6-OHDA处理BV2小胶质细胞,细胞的铁转出能力无明显变化。(P0.05)。4.10μmol/L 6-OHDA处理BV2小胶质细胞24h,与对照组相比,hepcidin的释放量明显下降,差异有统计学意义(P0.01)。5.10μmol/L的6-OHDA处理BV2小胶质细胞24h,TNF-αm RNA的水平显著升高,与正常对照组相比,差异有统计学意义(P0.01)。IL-1βm RNA的水平显著升高,与正常对照组相比,差别有统计学意义(P0.01)。6.100μmol/L FAC处理BV2小胶质细胞24h,DMT1蛋白表达明显降低,与对照组相比,差异有统计学意义(P0.01)。FPN1蛋白表达明显升高,与对照组相比,差异有统计学意义(P0.01)。7.100μmol/L FAC处理BV2小胶质细胞24h,IRP1的蛋白表达明显降低,与正常对照组相比,差异有统计学意义(P0.001)。8.100μmol/L DFO处理BV2小胶质细胞24h,DMT1蛋白表达明显升高,与对照组相比,差异有统计学意义(P0.01)。FPN1蛋白表达明显降低,与对照组相比,差异有统计学意义(P0.01)。9.100μmol/L DFO处理BV2小胶质细胞24h,IRP1的蛋白表达明显升高,与正常对照组相比,差异有统计学意义(P0.01)。上述结果表明,6-OHDA处理BV2小胶质细胞DMT1表达增加,铁转入增加,可能与IRP1上调有关。6-OHDA对FPN1表达无明显作用,铁转出不变,可能是IRP1上调,hepcidin下调共同作用的结果。6-OHDA能激活BV2小胶质细胞,使细胞TNF-α,IL-1β表达增加。高铁抑制了IRP1,使DMT1蛋白表达降低,FPN1的蛋白表达升高。低铁激活了IRP1,使DMT1蛋白表达升高,FPN1的蛋白表达降低。综上所述,6-OHDA通过激活IRP1,抑制hepcidin,调节了DMT1和FPN1的表达。引起小胶质细胞铁聚积。6-OHDA能激活小胶质细胞,释放炎性因子,进而造成DA神经元的损伤。
[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.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.5

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