本文選題：外泌體 + 帕金森病　； 參考：《中南大學(xué)》2014年博士論文

【摘要】：背景： 帕金森病(Parkinson's disease, PD)也稱震顫麻痹(Paralysis agitans)是發(fā)病率僅次于阿爾茨海默病的第二大神經(jīng)退行性疾病。臨床表現(xiàn)主要為運(yùn)動(dòng)遲緩、靜止性震顫、肌張力增高、姿勢(shì)步態(tài)異常及嗅覺(jué)減退、睡眠障礙等。帕金森病的病因和發(fā)病機(jī)制目前尚不明確,普遍認(rèn)為可能與遺傳因素、環(huán)境因素和老化等多種因素有關(guān)。帕金森病最重要的病理學(xué)改變是中腦黑質(zhì)(Substantia nigra, SN)多巴胺(Dopamine, DA)能神經(jīng)元變性缺失及殘存多巴胺能神經(jīng)元內(nèi)形成以α-突觸核蛋白(α-synuclein)為主要成分的嗜酸性包涵體,即路易氏小體(Lewy Body)。 外泌體(Exosomes)是一種直徑約30-100nm,密度范圍在1.13-1.19g/m1之間的膜性微囊泡,.電鏡下觀察形狀呈雙凹圓盤(pán)狀或杯口狀。細(xì)胞內(nèi)的多泡體(Multivesicular bodies, MVBs)和胞漿膜融合后釋放管腔內(nèi)囊泡(Intraluminal vesicles, ILVs),一旦其進(jìn)入細(xì)胞外環(huán)境中稱為外泌體,可以由體內(nèi)和體外培養(yǎng)的不同類型活細(xì)胞釋放,含有蛋白質(zhì)和RNA等成分。外泌體作為一種納米級(jí)的膜性囊泡于20世紀(jì)80年代末在羊網(wǎng)織紅細(xì)胞培養(yǎng)分化過(guò)程中首次被發(fā)現(xiàn),隨后Caby等于2005年首次在健康人群的體內(nèi)(血液)中檢測(cè)到外泌體,提示其作為一種分子媒介,促進(jìn)細(xì)胞間或器官間的信息交流。近些年來(lái),針對(duì)外泌體與疾病的關(guān)系進(jìn)行了大量研究,已證實(shí)其在阿爾茨海默病和朊蛋白病等中樞神經(jīng)系統(tǒng)疾病發(fā)病中起作用,外泌體可以作為一種新型的疾病標(biāo)記物,能夠在細(xì)胞間傳遞信息從而導(dǎo)致疾病發(fā)生。 如前所述,來(lái)源于多泡體的外泌體可與溶酶體融合而降解,或者與胞膜融合向細(xì)胞外釋放外泌體。一系列帕金森病相關(guān)基因,攜帶有致病突變(LRRK2、ATP13A2和VPS35)或者作為發(fā)病危險(xiǎn)因素的常見(jiàn)變異(GBA的雜合突變),被證實(shí)參與了溶酶體-內(nèi)吞途徑。最近的研究顯示LRRK2直接影響細(xì)胞分泌和內(nèi)吞機(jī)制,LRRK2還被發(fā)現(xiàn)與多泡體(MVBs)共定位(co-localize)。α-突觸核蛋白分泌后在腦脊液、血漿、唾液和細(xì)胞培養(yǎng)液中均能檢測(cè)到,Emmanouilidou等于2010年首次發(fā)現(xiàn)細(xì)胞產(chǎn)生的α-突觸核蛋白通過(guò)外泌體、鈣依賴機(jī)制分泌到胞外,隨后Alvarez等人也發(fā)現(xiàn)了類似現(xiàn)象,這一研究結(jié)果強(qiáng)調(diào)了外泌體在α-突觸核蛋白細(xì)胞間轉(zhuǎn)運(yùn)的作用,進(jìn)一步增強(qiáng)了外泌體與帕金森病發(fā)病機(jī)制的相關(guān)性,為以后帕金森病早期診斷和靶向治療提供理論基礎(chǔ)。 盡管大量實(shí)驗(yàn)已發(fā)現(xiàn)外泌體在正常人和腫瘤患者體液中表達(dá),目前尚未有關(guān)于帕金森病患者的外周血中外泌體的檢測(cè)研究。以往的研究發(fā)現(xiàn)α-突觸核蛋白存在于血清中,SH-SY5Y細(xì)胞系分泌的外泌體中也含有α-突觸核蛋白,目前尚沒(méi)有關(guān)于帕金森病患者的血清外泌體中是否存在α-突觸核蛋白的相關(guān)報(bào)道。目的： 探討帕金森病患者血清中是否存在外泌體,探索血清外泌體中是否表達(dá)帕金森病相關(guān)蛋白質(zhì)、其表達(dá)是否與帕金森病關(guān)聯(lián),進(jìn)一步利用外泌體為帕金森病的早期診斷和靶向治療提供理論依據(jù)。 方法： 收集帕金森病患者和性別年齡匹配的正常對(duì)照者血清樣本各20例,分別用差速離心法和ExoQuick試劑法從血清中分離提純外泌體；用透射電鏡鑒定兩組血清外泌體形態(tài)、western blot檢測(cè)兩組血清外泌體標(biāo)記蛋白CD63和CD9；并用western blot檢測(cè)兩組血清外泌體中是否表達(dá)α-突觸核蛋白和LRRK2蛋白。結(jié)果： 1.帕金森病患者外周血清中檢測(cè)到大小均一、直徑為30-100nm的膜性微囊泡結(jié)構(gòu),經(jīng)western blot證實(shí)其內(nèi)表達(dá)外泌體標(biāo)記蛋白CD63、CD9,鑒定即外泌體；ExoQuick試劑法分離收集的外泌體較差速離心法產(chǎn)量更高,濃度更純。 2.帕金森病患者血清外泌體中檢測(cè)到帕金森病相關(guān)蛋白α-突觸核蛋白的表達(dá),且其含量較正常對(duì)照者顯著減少(P=0.016),兩組血清外泌體中均未檢測(cè)到LRRK2蛋白表達(dá)。結(jié)論： 1.首次證實(shí)帕金森病患者外周血清中存在外泌體。 2.首次證實(shí)帕金森病患者外周血清外泌體中含有α-突觸核蛋白,且表達(dá)量較正常對(duì)照顯著減少,表明其與帕金森病存在關(guān)聯(lián)。 3.帕金森病者外周血清外泌體中可能不存在LRRK2蛋白,仍需要擴(kuò)大樣本進(jìn)一步驗(yàn)證。
[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.
Method錛，

本文編號(hào)：1895018