血清外泌体分离鉴定及其内含物与帕金森病关联研究

发布时间：2018-05-16 02:21

  本文选题：外泌体 + 帕金森病　； 参考：《中南大学》2014年博士论文

【摘要】：背景： 帕金森病(Parkinson's disease, PD)也称震颤麻痹(Paralysis agitans)是发病率仅次于阿尔茨海默病的第二大神经退行性疾病。临床表现主要为运动迟缓、静止性震颤、肌张力增高、姿势步态异常及嗅觉减退、睡眠障碍等。帕金森病的病因和发病机制目前尚不明确,普遍认为可能与遗传因素、环境因素和老化等多种因素有关。帕金森病最重要的病理学改变是中脑黑质(Substantia nigra, SN)多巴胺(Dopamine, DA)能神经元变性缺失及残存多巴胺能神经元内形成以α-突触核蛋白(α-synuclein)为主要成分的嗜酸性包涵体,即路易氏小体(Lewy Body)。 外泌体(Exosomes)是一种直径约30-100nm,密度范围在1.13-1.19g/m1之间的膜性微囊泡,.电镜下观察形状呈双凹圆盘状或杯口状。细胞内的多泡体(Multivesicular bodies, MVBs)和胞浆膜融合后释放管腔内囊泡(Intraluminal vesicles, ILVs),一旦其进入细胞外环境中称为外泌体,可以由体内和体外培养的不同类型活细胞释放,含有蛋白质和RNA等成分。外泌体作为一种纳米级的膜性囊泡于20世纪80年代末在羊网织红细胞培养分化过程中首次被发现,随后Caby等于2005年首次在健康人群的体内(血液)中检测到外泌体,提示其作为一种分子媒介,促进细胞间或器官间的信息交流。近些年来,针对外泌体与疾病的关系进行了大量研究,已证实其在阿尔茨海默病和朊蛋白病等中枢神经系统疾病发病中起作用,外泌体可以作为一种新型的疾病标记物,能够在细胞间传递信息从而导致疾病发生。 如前所述,来源于多泡体的外泌体可与溶酶体融合而降解,或者与胞膜融合向细胞外释放外泌体。一系列帕金森病相关基因,携带有致病突变(LRRK2、ATP13A2和VPS35)或者作为发病危险因素的常见变异(GBA的杂合突变),被证实参与了溶酶体-内吞途径。最近的研究显示LRRK2直接影响细胞分泌和内吞机制,LRRK2还被发现与多泡体(MVBs)共定位(co-localize)。α-突触核蛋白分泌后在脑脊液、血浆、唾液和细胞培养液中均能检测到,Emmanouilidou等于2010年首次发现细胞产生的α-突触核蛋白通过外泌体、钙依赖机制分泌到胞外,随后Alvarez等人也发现了类似现象,这一研究结果强调了外泌体在α-突触核蛋白细胞间转运的作用,进一步增强了外泌体与帕金森病发病机制的相关性,为以后帕金森病早期诊断和靶向治疗提供理论基础。 尽管大量实验已发现外泌体在正常人和肿瘤患者体液中表达,目前尚未有关于帕金森病患者的外周血中外泌体的检测研究。以往的研究发现α-突触核蛋白存在于血清中,SH-SY5Y细胞系分泌的外泌体中也含有α-突触核蛋白,目前尚没有关于帕金森病患者的血清外泌体中是否存在α-突触核蛋白的相关报道。目的： 探讨帕金森病患者血清中是否存在外泌体,探索血清外泌体中是否表达帕金森病相关蛋白质、其表达是否与帕金森病关联,进一步利用外泌体为帕金森病的早期诊断和靶向治疗提供理论依据。 方法： 收集帕金森病患者和性别年龄匹配的正常对照者血清样本各20例,分别用差速离心法和ExoQuick试剂法从血清中分离提纯外泌体；用透射电镜鉴定两组血清外泌体形态、western blot检测两组血清外泌体标记蛋白CD63和CD9；并用western blot检测两组血清外泌体中是否表达α-突触核蛋白和LRRK2蛋白。结果： 1.帕金森病患者外周血清中检测到大小均一、直径为30-100nm的膜性微囊泡结构,经western blot证实其内表达外泌体标记蛋白CD63、CD9,鉴定即外泌体；ExoQuick试剂法分离收集的外泌体较差速离心法产量更高,浓度更纯。 2.帕金森病患者血清外泌体中检测到帕金森病相关蛋白α-突触核蛋白的表达,且其含量较正常对照者显著减少(P=0.016),两组血清外泌体中均未检测到LRRK2蛋白表达。结论： 1.首次证实帕金森病患者外周血清中存在外泌体。 2.首次证实帕金森病患者外周血清外泌体中含有α-突触核蛋白,且表达量较正常对照显著减少,表明其与帕金森病存在关联。 3.帕金森病者外周血清外泌体中可能不存在LRRK2蛋白,仍需要扩大样本进一步验证。
[Abstract]:Background:
Parkinson's disease (Parkinson's disease, PD), also known as Paralysis agitans (Paralysis agitans), is the second largest neurodegenerative disease after Alzheimer's disease. The clinical manifestations are mainly kinesic, static tremor, muscle tension, abnormal postural gait and olfactory impairment, sleep disorder, and so on. The etiology and pathogenesis of Parkinson's disease The most important pathological changes of Parkinson's disease are the degeneration of Substantia nigra (SN) dopamine (Dopamine, DA) and the formation of alpha synuclein (alpha -synuclein) in the remnant dopaminergic neurons of the mesencephalic acid (Dopamine, DA). The eosinophilic inclusion body is called Lewy Body.
Exocrine (Exosomes) is a membranous microvesicle with a diameter of about 30-100nm and a density range of 1.13-1.19g/m1. Under electron microscope, the shape of the membrane is a double concave disk or a cup. The intracellular vesicles (Multivesicular bodies, MVBs) and the cytoplasm membrane fusion release the inner cavity vesicles (Intraluminal vesicles, ILVs), once they enter the cells. The environment, called exocrine, can be released by different types of living cells in vivo and in vitro, containing proteins and RNA. Exosbodies were first discovered in the process of culture and differentiation of sheep reticulocytes in the end of 1980s as a kind of nanoscale membranous vesicles, and then Caby was the first in the body of healthy people (blood) in 2005. The exocrine was detected in the liquid, suggesting that it was used as a molecular medium to promote the exchange of information between the cells and the organs. In recent years, a lot of studies have been conducted on the relationship between the external secretory and the disease. It has been proved that it plays a role in Alzheimer's disease and prion disease and other central nervous system diseases, and the exocrine can be used as a new type of disease. Disease markers can transmit information between cells, leading to disease.
As mentioned earlier, the exosomes derived from the multivesicles can be degraded with the lysosomes, or release exosomes from the cell membrane. A series of Parkinson disease related genes, which carry a pathogenic mutation (LRRK2, ATP13A2, and VPS35), or as a common variation of the risk factors (GBA heterozygous mutation), have been confirmed to be involved in the lysosome. Endocytosis. Recent studies have shown that LRRK2 directly affects cell secretion and endocytosis, and LRRK2 is also found to co localize (co-localize) with multiple vesicles (MVBs). Alpha synuclein is detected in cerebrospinal fluid, plasma, saliva and cell culture, and Emmanouilidou equals the first discovery of alpha synuclein produced by cells in 2010. Over exocrine, the calcium dependence mechanism is secreted to the extracellular, and then Alvarez and others have found a similar phenomenon. The results emphasize the role of the exocrine intercellular transport in the alpha synuclein cells and further enhance the correlation between the exocrine and the pathogenesis of Parkinson's disease, providing a theory for the early diagnosis and targeted therapy of post Parkinson disease. Basics.
Although a large number of experiments have been found to express the exocrine in the body fluids of normal and tumor patients, there is no study on the detection of foreign secretions in peripheral blood of patients with Parkinson's disease. Previous studies have found that alpha synuclein exists in serum, and the exocrine secreted by SH-SY5Y cell lines also contain alpha synuclein, which is not yet concerned. Objective: to report the presence of alpha synuclein in the serum exudate of patients with Parkinson's disease.
To explore the presence of exocrine in the serum of patients with Parkinson's disease and to explore whether the expression of Parkinson's disease related proteins in the serum exocrine is related to Parkinson's disease and to provide a theoretical basis for the early diagnosis and target therapy of Parkinson disease.
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