环境应答基因JWA拮抗帕金森病发生的作用及机制研究

发布时间：2018-09-18 14:45

【摘要】：帕金森病为仅次于阿尔兹海默病的人类第二大神经退行性病变。帕金森病的发病率伴随着年龄的增加而增加,60岁以上的人群中发病率为1-4%。主要的临床表现有肌肉僵直、震颤、运动迟缓以及姿势不稳定等。帕金森病的病理特征主要有多巴胺能神经元的丢失,路易小体的沉积以及胶质细胞的活化等等。尽管目前帕金森病的发病原因不明,但主要认为与衰老、氧化应激、炎症反应、DNA损伤等等因素有关。临床上,左旋多巴是治疗帕金森病的一线药物,但仅能改善症状并不能逆转帕金森病的发生、发展。目前,寻找新的帕金森病治疗靶标、研究新型治疗药物十分紧迫和必要。帕金森病人脑片上不仅发现有多巴胺能神经元的变性、坏死还有星形胶质细胞大量的活化、增生。星形胶质细胞参与了多种包括帕金森病在内的神经退行性疾病。星形胶质细胞在维持中枢神经系统稳定上发挥了重要作用,包括突触谷胱甘肽的摄取、细胞内外K离子水平的稳定以及提供神经元多种神经营养因子等。环境应答基因JWA,是一种新的微管结合蛋白,通过MAPK信号通路调节癌症细胞的迁移以及介导白血病细胞的分化。JWA通过调控XRCC1的表达参与DNA损伤后的BER修复通路,参与氧化应激诱导的DNA损伤修复。JWA不仅参与氧化还原平衡且参与调节炎症反应。虽然JWA发挥着多样作用,但是其在帕金森病方面研究目前还没有相关报道。本研究中我们主要从构建条件性星形胶质细胞JWA剔除小鼠入手,研究星形胶质细胞中JWA对帕金森病多巴胺能神经元的影响。目的：在整体动物、细胞和分子水平探讨星形胶质细胞中JWA的表达对帕金森病多巴胺能神经元功能的影响及机制。方法：1、繁殖3月龄雄性C57BL/6J野生型小鼠(JWAloxp/loxp)及条件性星形胶质细胞JWA剔除小鼠(JWAΔ2/Δ2/GFAP-Cre)。2、应用酪氨酸羟化酶免疫组织化学结合体视学计数分析黑质致密部多巴胺能神经元的损伤,同时应用尼氏染色结合体视学计数分析中脑神经元的损伤；检测中脑黑质致密部星形胶质细胞的活化。3、运用高效液相色谱法测定单胺类神经递质含量。4、运用免疫荧光双标来检测中脑多巴胺能神经元中活性氧的含量以及运用流式检测给予MPP+50μM24h人星形胶质瘤细胞(U251细胞)内活性氧含量的变化。5、应用谷胱甘肽试剂盒检测给予MPP+50μM24h后谷胱甘肽的含量。6、应用Western-blotting法来检测给予MPP+50μM24h后U251细胞的Iκβ、IκB、胞浆胞核p65分子含量变化以及检测中脑组织中Ikkβ、IκB、胞浆胞核p65的分子含量变化。结果：1. JWAΔ2/Δ2/GFAP-Cre小鼠导致中脑黑质致密部星形胶质细胞显著活化(1.34×103±74.5vs.8.44×103±1.35×103, P0.01)并伴有多巴胺能神经元的丢失(1.03×104±238vs.6.17×103±392, P0.001)。2. JWAΔ2/Δ2/GFAP-Cre小鼠多巴胺含量显著升高(11.1±1.79nmol/g vs.22.5±2.23nmol/g, P0.01)。但是DOPAC/DA(3.28±0.414vs.2.02±0.285, P0.05)以及HVA(13.8±0.742nmol/g vs.10.6±0.870nmol/g, P0.05)相对于对照小鼠显著降低。3.JWA缺乏显著加剧中脑多巴胺能神经元活性氧的聚积。在U251细胞中增加JWA的表达可以显著抑制活性氧水平(3.60×104±572vs2.87×104±397,P0.001)且显著增加谷胱甘肽的含量(298±0.667vs.365±7.09, P0.001)。4. JWAΔ2/Δ2/GFAP-Cre小鼠中脑组织细胞核内p65的表达是对照小鼠JWALoxp/Loxp的1.76倍(P0.01)；IKKβ的含量是对照小鼠的1.7倍(P0.05),而IκκC的表达是对照小鼠的0.5倍(P0.001)。在体外模型中,给予U251细胞MPP+(50μM)24h后,高表达JWA的U251细胞核内p65表达量是对照组的70%(P0.001),而胞浆p65的表达量是对照组的2.1倍(P0.01)；高表达JWA组IκκC的表达是对照组的1.7倍,IKKβ和p-p65的表达分别是对照组的0.6和0.5倍(P0.001)。结论：环境应答基因拮抗PD的发生的作用以及机制研究：1.条件性星形胶质细胞JWA基因剔除(JWA△2/△2／GFAP-Cre)小鼠星形胶质细胞活化且促进多巴胺能神经元的丢失,JWA基因在体内体外模型中对多巴胺能神经元起保护作用。2.条件性星形胶质细胞JWA基因剔除通过JWA-IKKβ-NF-κB信号通路导致多巴胺能神经元的损伤。
[Abstract]:Parkinson's disease is the second largest neurodegenerative disorder in humans after Alzheimer's disease. The incidence of Parkinson's disease increases with age. The incidence of Parkinson's disease is 1-4% in people over the age of 60. The main clinical manifestations are muscle stiffness, tremor, motor retardation and postural instability. Loss of dopaminergic neurons, deposition of Lewis bodies, activation of glial cells and so on. Although the etiology of Parkinson's disease is unknown, it is mainly associated with aging, oxidative stress, inflammation, DNA damage and other factors. It can reverse the occurrence and development of Parkinson's disease. At present, it is very urgent and necessary to find new targets for the treatment of Parkinson's disease and study new therapeutic drugs.
In Parkinson's disease, not only degeneration of dopaminergic neurons, necrosis, but also activation and proliferation of astrocytes have been found. Astrocytes are involved in a variety of neurodegenerative diseases including Parkinson's disease. Astrocytes play an important role in maintaining the stability of the central nervous system, including synaptic glutathione. The uptake of glycine, the stability of intracellular and extracellular K ions, and the availability of various neurotrophic factors in neurons.
JWA, a novel microtubule-binding protein, regulates the migration of cancer cells and mediates the differentiation of leukemia cells through MAPK signaling. JWA participates in the BER repair pathway after DNA damage by regulating the expression of XRCC1. JWA participates in the repair of DNA damage induced by oxidative stress. Inflammation. Although JWA plays a variety of roles, its research on Parkinson's disease has not been reported. In this study, we mainly constructed conditioned astrocyte JWA knockout mice to study the effect of JWA on dopaminergic neurons in Parkinson's disease.
AIM: To investigate the effect of JWA expression in astrocytes on the function of dopaminergic neurons in Parkinson's disease at the animal, cellular and molecular levels.
METHODS: 1. Three-month-old male C57BL/6J wild-type mice (JWA loxp/loxp) and conditioned astrocyte JWA knockout mice (JWA 2/2/GFAP-Cre). 2. The damage of dopaminergic neurons in substantia nigra compact was analyzed by tyrosine hydroxylase immunohistochemical binding stereological counting and Nissl's staining binding stereological counting. To analyze the damage of mesencephalic neurons; to detect the activation of astrocytes in the compact part of substantia nigra. 3. To determine the content of monoamine neurotransmitters by high performance liquid chromatography. 4. To detect the content of reactive oxygen species in mesencephalic dopaminergic neurons by immunofluorescence double labeling and to detect the content of MPP + 50 mu M24 h human astrocytoma cells by flow cytometry. 5. Glutathione assay kit was used to detect the content of glutathione. 6. Western-blotting was used to detect the content of I-kappa beta, I-kappa B, p65 in nucleus and Ikk-beta, I-kappa B in mesencephalic tissue. Change.
Results: 1. JWA2/2/GFAP-Cre mice significantly activated astrocytes in the substantia nigra compacta (1.34 *103 +74.5 vs. 8.44 *103 +1.35 *103, P However, DOPAC/DA (3.28+0.414 vs.2.02+0.285, P 0.05) and HVA (13.8+0.742 nmol/g vs.10.6+0.870 nmol/g, P 0.05) decreased significantly compared with the control mice. 3. JWA deficiency significantly increased the accumulation of reactive oxygen species in mesencephalic dopaminergic neurons. Increasing the expression of JWA in U251 cells significantly inhibited the accumulation of reactive oxygen species (ROS). The expression of p65 in the nucleus of JWA2/2/GFAP-Cre mice was 1.76 times as high as that of JWALoxp/Loxp (P 0.01), the content of IKK beta was 1.7 times as high as that of control mice (P 0.05), and the expression of Ikappa C was 1.76 times as high as that of control mice (P 0.05). The expression of p65 in U251 nucleus was 70% (P 0.001) and that in cytoplasm was 2.1 times (P 0.01) of the control group. The expression of Ikappa-kappa C was 1.7 times of the control group, and the expression of IKK-beta and p-p65 were 0.6 and 0.6 times of the control group, respectively. 5 times (P0.001).
CONCLUSIONS: Environmental response genes antagonize the development of PD and its mechanism: 1. Conditioned astrocyte JWA gene knockout (JWA {2 / {2}/ GFAP-Cre} mice astrocytes activate and promote the loss of dopaminergic neurons. JWA gene plays a protective role on dopaminergic neurons in vitro and in vivo. 2. JWA gene knockout in astrocytes results in damage to dopaminergic neurons via JWA-IKK beta-NF-kappa B signaling pathway.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5

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