六种不同组织来源间充质干细胞对阿茨海默症模型中损伤神经细胞的修复作用

发布时间：2018-09-01 10:12

【摘要】：研究背景和目的:阿尔茨海默症(Alzheimer' s disease,AD)是一种发病原因不明的进行性神经退行性疾病,该病的主要表现为全脑不可逆转性脑功能损害,是公认的一种难治性疾病,其发病的主要早期症状是近期记忆的障碍,并且其发病率和年龄的增长密切相关。根据世界阿尔茨海默症报道,2015年全球对AD患者四千六百八十万,花费达八千一百八十万亿美元,预计到2030年,AD患者的人数将达到七千一百七十万人,势必给社会和家庭带来更为沉重的经济负担,也严重影响到了患者家庭的生活质量。目前临床上针对AD的治疗方法主要包括药物治疗和非药物治疗,但大多都仅仅是对症治疗,不能有效治愈疾病,也难以阻止疾病的进一步发展。因此,研发新的药物或者策略,用于治疗或者预防AD,改善患者的生活质量,就至关重要。目前针对AD的研究中,干细胞逐渐受到越来越多的关注,近年来科学家们针对干细胞治疗AD开展的基础和临床研究,结果表明干细胞移植有望对AD等多种神经相关疾病具有潜在的治疗作用。由于干细胞本身所具有的高度增殖能力、多向分化潜能、旁分泌多种活性因子参与细胞存活、发育分化、微环境改善、抗凋亡、促血管新生等特点和优势,以及其展现出的良好疗效和临床应用的可行性,使其有望在临床AD的治疗中发挥重要作用。在不同的干细胞中,综合其来源、安全性、临床应用、治疗效果、伦理、免疫等方面的考虑,间充质干细胞(MSCs)就展现出其应用安全和有效的优势。MSCs的来源多样,可以从牙齿、脐带、胎盘、羊水、骨髓、脂肪等组织中获取;具有较强的多向分化能力,可以向多种组织细胞分化,甚至还可以向神经元分化;此外,其强大的分泌作用可调节免疫反应、发挥营养支持作用。但在这些研究中,不同来源的MSCs并未在同一种疾病、相同体外培养及评价体系中进行综合性评价研究,难以得出准确的治疗效果评价,也未能阐明其可能的作用机制。本研究的目的为:评价多种年轻组织来源的MSCs在AD细胞模型中修复损伤神经细胞的效果,并对其可能的作用机制进行探讨。方法:本研究采用经典的原代分离方法制备牙周膜、牙髓、脐带、羊膜、平滑绒毛膜、骨髓六种不同组织来源的间充质干细胞,对所制备的MSCs进行鉴定。与此同时,我们选取冈田酸(OA)作用于神经母细胞瘤细胞系(SH-SY5Y)和原代神经元,模拟神经纤维缠结这一病理特征,建立AD细胞模型。在此基础上,收集不同来源的MSCs细胞的条件培养基,与已经成功建立的体外AD模型细胞进行共培养,并从细胞形态、细胞活力和增殖能力、线粒体功能、树突棘数量、超微结构等方面评估MSCs对该细胞模型的治疗作用。进一步利用Western Blot方法,在蛋白水平检测与AD发生发展密切相关的Tau蛋白396位点丝氨酸磷酸化的表达情况,同时利用实时定量PCR的方法筛选了多种与认知相关基因的表达。结果:SH-SY5Y细胞和原代神经元经过OA处理后,胞体皱缩、变圆,树突回缩、变短,神经细胞树突的平均长度缩短了 66.6%;SH-SY5Y细胞活力下降了35%,线粒体膜电位降低了 13.9%,神经元树突棘数量减少了 64.4%,细胞骨架排列紊乱,Tau蛋白磷酸化396位点上调,结果证明我们成功建立了 AD细胞模型。在此基础上,我们利用MSCs治疗经过OA损伤的神经细胞,发现所有经过MSCs治疗后的神经细胞形态恢复,树突伸出、变长,树突平均长度增加了 94%,细胞活力增强了10%,线粒体膜电位升高了 6.4%以上,神经元树突棘增加了一倍以上,骨架排列整齐。通过Western Blot检测发现经治疗后的Tau蛋白Ser396磷酸化的表达水平降低了 35%以上,而BRCA1基因的表达量均较OA组明显升高,增加了 116%以上,提示MSCs可能是通过降低Tau蛋白的磷酸化以及上调BRCA1这一基因的表达来发挥其治疗受损神经细胞的作用。总结:目前研究国内外主要针对脐带、脂肪等组织来源的间充质干细胞进行研究,但并未有文献报道牙周膜和平滑绒毛膜来源的间充质干细胞对AD的研究,且没有将多种组织来源的间充质干细胞在同一疾病、同一模型中进行综合性的评估证明六种不同组织来源的MSCs能够通过其分泌的因子发挥对OA损伤神经细胞的治疗作用,而且首次提出MSCs其可能的作用机制是通过降低受损神经细胞中Tau蛋白的过度磷酸化和激活神经细胞中BRCA1的表达而发挥作用的。
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

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