【摘要】：研究背景和目的:阿爾茨海默癥(Alzheimer' s disease,AD)是一種發(fā)病原因不明的進(jìn)行性神經(jīng)退行性疾病,該病的主要表現(xiàn)為全腦不可逆轉(zhuǎn)性腦功能損害,是公認(rèn)的一種難治性疾病,其發(fā)病的主要早期癥狀是近期記憶的障礙,并且其發(fā)病率和年齡的增長密切相關(guān)。根據(jù)世界阿爾茨海默癥報(bào)道,2015年全球?qū)D患者四千六百八十萬,花費(fèi)達(dá)八千一百八十萬億美元,預(yù)計(jì)到2030年,AD患者的人數(shù)將達(dá)到七千一百七十萬人,勢(shì)必給社會(huì)和家庭帶來更為沉重的經(jīng)濟(jì)負(fù)擔(dān),也嚴(yán)重影響到了患者家庭的生活質(zhì)量。目前臨床上針對(duì)AD的治療方法主要包括藥物治療和非藥物治療,但大多都僅僅是對(duì)癥治療,不能有效治愈疾病,也難以阻止疾病的進(jìn)一步發(fā)展。因此,研發(fā)新的藥物或者策略,用于治療或者預(yù)防AD,改善患者的生活質(zhì)量,就至關(guān)重要。目前針對(duì)AD的研究中,干細(xì)胞逐漸受到越來越多的關(guān)注,近年來科學(xué)家們針對(duì)干細(xì)胞治療AD開展的基礎(chǔ)和臨床研究,結(jié)果表明干細(xì)胞移植有望對(duì)AD等多種神經(jīng)相關(guān)疾病具有潛在的治療作用。由于干細(xì)胞本身所具有的高度增殖能力、多向分化潛能、旁分泌多種活性因子參與細(xì)胞存活、發(fā)育分化、微環(huán)境改善、抗凋亡、促血管新生等特點(diǎn)和優(yōu)勢(shì),以及其展現(xiàn)出的良好療效和臨床應(yīng)用的可行性,使其有望在臨床AD的治療中發(fā)揮重要作用。在不同的干細(xì)胞中,綜合其來源、安全性、臨床應(yīng)用、治療效果、倫理、免疫等方面的考慮,間充質(zhì)干細(xì)胞(MSCs)就展現(xiàn)出其應(yīng)用安全和有效的優(yōu)勢(shì)。MSCs的來源多樣,可以從牙齒、臍帶、胎盤、羊水、骨髓、脂肪等組織中獲取;具有較強(qiáng)的多向分化能力,可以向多種組織細(xì)胞分化,甚至還可以向神經(jīng)元分化;此外,其強(qiáng)大的分泌作用可調(diào)節(jié)免疫反應(yīng)、發(fā)揮營養(yǎng)支持作用。但在這些研究中,不同來源的MSCs并未在同一種疾病、相同體外培養(yǎng)及評(píng)價(jià)體系中進(jìn)行綜合性評(píng)價(jià)研究,難以得出準(zhǔn)確的治療效果評(píng)價(jià),也未能闡明其可能的作用機(jī)制。本研究的目的為:評(píng)價(jià)多種年輕組織來源的MSCs在AD細(xì)胞模型中修復(fù)損傷神經(jīng)細(xì)胞的效果,并對(duì)其可能的作用機(jī)制進(jìn)行探討。方法:本研究采用經(jīng)典的原代分離方法制備牙周膜、牙髓、臍帶、羊膜、平滑絨毛膜、骨髓六種不同組織來源的間充質(zhì)干細(xì)胞,對(duì)所制備的MSCs進(jìn)行鑒定。與此同時(shí),我們選取岡田酸(OA)作用于神經(jīng)母細(xì)胞瘤細(xì)胞系(SH-SY5Y)和原代神經(jīng)元,模擬神經(jīng)纖維纏結(jié)這一病理特征,建立AD細(xì)胞模型。在此基礎(chǔ)上,收集不同來源的MSCs細(xì)胞的條件培養(yǎng)基,與已經(jīng)成功建立的體外AD模型細(xì)胞進(jìn)行共培養(yǎng),并從細(xì)胞形態(tài)、細(xì)胞活力和增殖能力、線粒體功能、樹突棘數(shù)量、超微結(jié)構(gòu)等方面評(píng)估MSCs對(duì)該細(xì)胞模型的治療作用。進(jìn)一步利用Western Blot方法,在蛋白水平檢測(cè)與AD發(fā)生發(fā)展密切相關(guān)的Tau蛋白396位點(diǎn)絲氨酸磷酸化的表達(dá)情況,同時(shí)利用實(shí)時(shí)定量PCR的方法篩選了多種與認(rèn)知相關(guān)基因的表達(dá)。結(jié)果:SH-SY5Y細(xì)胞和原代神經(jīng)元經(jīng)過OA處理后,胞體皺縮、變圓,樹突回縮、變短,神經(jīng)細(xì)胞樹突的平均長度縮短了 66.6%;SH-SY5Y細(xì)胞活力下降了35%,線粒體膜電位降低了 13.9%,神經(jīng)元樹突棘數(shù)量減少了 64.4%,細(xì)胞骨架排列紊亂,Tau蛋白磷酸化396位點(diǎn)上調(diào),結(jié)果證明我們成功建立了 AD細(xì)胞模型。在此基礎(chǔ)上,我們利用MSCs治療經(jīng)過OA損傷的神經(jīng)細(xì)胞,發(fā)現(xiàn)所有經(jīng)過MSCs治療后的神經(jīng)細(xì)胞形態(tài)恢復(fù),樹突伸出、變長,樹突平均長度增加了 94%,細(xì)胞活力增強(qiáng)了10%,線粒體膜電位升高了 6.4%以上,神經(jīng)元樹突棘增加了一倍以上,骨架排列整齊。通過Western Blot檢測(cè)發(fā)現(xiàn)經(jīng)治療后的Tau蛋白Ser396磷酸化的表達(dá)水平降低了 35%以上,而BRCA1基因的表達(dá)量均較OA組明顯升高,增加了 116%以上,提示MSCs可能是通過降低Tau蛋白的磷酸化以及上調(diào)BRCA1這一基因的表達(dá)來發(fā)揮其治療受損神經(jīng)細(xì)胞的作用�？偨Y(jié):目前研究國內(nèi)外主要針對(duì)臍帶、脂肪等組織來源的間充質(zhì)干細(xì)胞進(jìn)行研究,但并未有文獻(xiàn)報(bào)道牙周膜和平滑絨毛膜來源的間充質(zhì)干細(xì)胞對(duì)AD的研究,且沒有將多種組織來源的間充質(zhì)干細(xì)胞在同一疾病、同一模型中進(jìn)行綜合性的評(píng)估證明六種不同組織來源的MSCs能夠通過其分泌的因子發(fā)揮對(duì)OA損傷神經(jīng)細(xì)胞的治療作用,而且首次提出MSCs其可能的作用機(jī)制是通過降低受損神經(jīng)細(xì)胞中Tau蛋白的過度磷酸化和激活神經(jīng)細(xì)胞中BRCA1的表達(dá)而發(fā)揮作用的。
[Abstract]:BACKGROUND AND OBJECTIVE: Alzheimer's disease (AD) is a progressive neurodegenerative disorder with unknown etiology. The main manifestation of AD is irreversible impairment of brain function, which is recognized as a refractory disease. The main early symptom of AD is short-term memory impairment, and its morbidity and incidence. According to the World Alzheimer's Disease Report, AD patients worldwide will be 46.8 million in 2015, spending $81.80 trillion. It is estimated that by 2030, the number of AD patients will reach 71.7 million, which is bound to bring a heavier economic burden to society and families, as well as a serious impact. At present, the treatment methods for AD mainly include drug therapy and non-drug therapy, but most of them are only symptomatic treatment, which can not effectively cure the disease and prevent the further development of the disease. At present, stem cells have attracted more and more attention in the research of AD. In recent years, scientists have carried out basic and clinical research on stem cell therapy for AD. The results show that stem cell transplantation is expected to have potential therapeutic effects on many neurological diseases such as AD. Proliferative ability, multidirectional differentiation potential, paracrine multiple active factors involved in cell survival, development and differentiation, microenvironment improvement, anti-apoptosis, promoting angiogenesis and other characteristics and advantages, as well as its good efficacy and clinical feasibility, so that it is expected to play an important role in the treatment of clinical AD. Mesenchymal stem cells (MSCs) can be obtained from various tissues, such as teeth, umbilical cord, placenta, amniotic fluid, bone marrow, fat and so on. They have strong multi-directional differentiation ability and can be used to many kinds of tissue cells. Differentiation can even differentiate into neurons; in addition, its strong secretion can regulate immune response and play a role of nutritional support. However, in these studies, MSCs from different sources have not been in the same disease, the same in vitro culture and evaluation system for comprehensive evaluation, it is difficult to obtain accurate evaluation of therapeutic effect, nor can it be achieved. To elucidate the possible mechanism of action, the aim of this study was to evaluate the effect of MSCs derived from various young tissues on repairing injured nerve cells in AD cell models and to explore the possible mechanism of action. At the same time, we selected Okadaic acid (OA) to act on neuroblastoma cell line (SH-SY5Y) and primary neurons to simulate the pathological characteristics of neurofibrillary tangles and establish AD cell model. On this basis, we collected the conditions of MSCs from different sources. The therapeutic effects of MSCs on AD model cells were evaluated in terms of cell morphology, cell viability and proliferation, mitochondrial function, number of dendritic spines and ultrastructure. Results: After OA treatment, SH-SY5Y cells and primary neurons shrank, rounded, dendrites shrank, shortened, and the average length of dendrites shortened by 66.6%; SH-SY5Y cells survived. The force decreased by 35%, the mitochondrial membrane potential decreased by 13.9%, the number of dendritic spines decreased by 64.4%, the cytoskeleton arranged disorderly, and the Tau protein phosphorylation 396 site up-regulated. The results showed that we successfully established the AD cell model. On this basis, we used MSCs to treat OA-injured neurons and found that all the neurons were treated with MSCs. The average length of dendrites increased by 94%, cell viability increased by 10%, mitochondrial membrane potential increased by more than 6.4%, dendritic spines of neurons more than doubled, and the skeleton arranged neatly. The expression of BRCA1 gene increased by 116% compared with OA group, suggesting that MSCs may play a role in the treatment of damaged nerve cells by decreasing the phosphorylation of Tau protein and up-regulating the expression of BRCA1 gene. Cell research, but there is no literature on the study of periodontal ligament and smooth chorion-derived mesenchymal stem cells on AD, and there is no multiple tissue-derived mesenchymal stem cells in the same disease, the same model for a comprehensive evaluation proved that six different tissue-derived MSCs can play a role in OA damage through its secreted factors. The therapeutic effect of MSCs on injured nerve cells was firstly proposed. The possible mechanism of MSCs was to reduce the hyperphosphorylation of Tau protein in injured nerve cells and activate the expression of BRCA1 in injured nerve cells.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R749.16

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 Yufang Yan;Tuo Ma;Kai Gong;Qiang Ao;Xiufang Zhang;Yao Gong;;Adipose-derived mesenchymal stem cell transplantation promotes adult neurogenesis in the brains of Alzheimer's disease mice[J];Neural Regeneration Research;2014年08期

2 Bo Song;Qiang Ao;Ying Niu;Qin Shen;Huancong Zuo;Xiufang Zhang;Yandao Gong;;Amyloid beta-peptide worsens cognitive impairment following cerebral ischemia-reperfusion injury[J];Neural Regeneration Research;2013年26期

，

本文編號(hào)：2216879