不同分子量和硫酸化程度肝素组分的抗阿尔茨海默病活性研究
发布时间：2018-09-14 10:47
【摘要】：肝素一直以来仅作为抗凝和抗血栓药物用于临床,作为一种糖胺聚糖,其结构特点是高分子和高负电密度；从已经阐明的抗凝活性看,其活性是分子量和硫酸基所依赖的。近年来,肝素的抗炎、抗补体激活等非抗凝活性也得到关注,但尚未得到应用。阿尔茨海默病(Alzheimer's disease, AD),又称早老性痴呆症,是一种神经系统退行性疾病,对老年人的健康产生极大威胁。AD的特征性病理学改变为大脑皮质神经细胞内神经原纤维缠结、细胞外大量老年斑形成和大脑皮质细胞减少。p-淀粉样蛋白(β-amyloid, AD)沉积被认为是老年斑的主要成分,也是阿尔茨海默病的发病机制之一。Ap的沉积被认为是由淀粉样前体蛋白(amyloid precursor protein, APP)的代谢异常导致的。寡聚化的Ap和Ap沉积物又会引发对脑组织的一系列损伤,最终导致AD病变。其中,神经炎症,特别是对脑内补体系统的激活,是Ap的重要损伤机制之一。已有研究发现,肝素对APP代谢相关酶有着重要作用,对AD引起的神经炎症也存在着重要影响,但缺乏系统和深入的研究。本课题利用Ap损伤的神经元样细胞作为细胞模型,研究不同分子量和硫酸化程度肝素组分对APP代谢和AD引发的补体激活的作用,以阐明肝素结构与其抗AD活性的关系。 本课题的研究内容及成果主要包括五个方面： 1AD细胞模型的构建 通过RA诱导分化人神经母细胞瘤细胞SH-SY5Y构建神经元样细胞模型。RA处理使SH-SY5Y的形态发生明显变化,形成神经元细胞特有的轴突的结构和细胞标记。利用MTT比色法测定Aβ1-42对神经元样细胞的损伤。确定用浓度为100μg/mL的Aβ1-42处理神经元样细胞24h可复制AD样细胞模型。 2不同分子量和硫酸化程度肝素组分对神经元样细胞和AD样细胞存活率的研究 利用MTT比色法测定了不同分子量和硫酸化程度的肝素组分对神经元样细胞和AD样细胞存活率的影响,包括肝素、依诺肝素、2-O-脱硫酸肝素、6-O-脱硫酸肝素、N-脱硫酸-乙酰化肝素、肝素二糖、肝素四糖、肝素十糖和肝素十六糖。不同肝素组分对神经元样细胞存活率没有显著性影响。而对于AD样细胞,分子量在3000-5000Da,且含有O-硫酸基和N-硫酸基的肝素组分,如依诺肝素、肝素十六糖等,能显著提高AD样细胞的存活率、抑制Ap对神经元样细胞的损伤的活性在所有肝素组分中。 3不同分子量和硫酸化程度肝素组分对APP代谢作用的研究 首次利用Ap损伤的神经元样细胞作为AD细胞模型,分析了Aβ1-42对神经元样细胞的APP代谢关键酶、APP和生成的相关片段的影响,以及肝素及其衍生物对Aβ1-42引起的上述变化的影响。Aβ1-42显著提高了BACE1的表达量,而肝素及其衍生物对其具有抑制作用,依诺肝素、肝素十糖和肝素十六糖对BACE-1生成的抑制作用最强。Aβ1-42提高了ADAM10前体及ADAM10表达量,但并不显著。结果提示肝素的硫酸基,特别是6-O-硫酸基和N-硫酸基,分子量在十糖到十六糖长度的糖链对肝素抑制Ap引起的APP代谢关键酶表达是必需的。 Aβ1-42提高了AD样细胞的APP表达量,降低了C99的表达量,而C83的表达量基本不变,但这些影响并不显著。2-O-脱硫酸肝素和肝素二糖、四糖、十糖、十六糖使C83表达量明显增加,提示肝素对ADAM10的酶活力可能存在着重要影响。 4不同分子量和硫酸化程度肝素组分对补体系统作用的研究 免疫荧光检查发现,Aβ1-42促进神经元样细胞表达C5b-9,而且封闭CD59对表达量并无明显影响。肝素能抑制Aβ1-42引起的C5b-9表达。 首次利用Aβ1-42损伤的神经元样细胞作为AD细胞模型,通过Wlestem b10tting分析了神经元样细胞细胞中不同系统相关蛋白的表达情况。Aβ1-42显著促进了C4a的表达,而肝素及其衍生物对C4a表达的抑制作用与肝素的硫酸基及分子量密切相关。6-O-硫酸基和N-硫酸基对于肝素抑制C4a表达的活性是必需的。十六糖或以上的肝素糖链才可能具有抑制C4a表达的活性。 Aβ1-42也能显著提高C3的表达量,肝素及其衍生物对其的抑制作用不具有显著性影响。 以上结果表明,肝素可通过调节APP代谢和Ap引起的补体系统激活抑制Aβ引起的对神经元样细胞的损伤,且这种作用都与肝素的硫酸化程度、硫酸基位置和分子量有关。从调节APP代谢和抑制补体激活的角度来看,具有抗AD活性的肝素应为十糖到十六糖的肝素寡糖,并具有所有的硫酸基,包括O-硫酸基和N-硫酸基。
[Abstract]:Heparin has been used only as anticoagulant and antithrombotic drugs in clinic. As a glycosaminoglycan, its structure is characterized by high molecular weight and high negative electric density. The anticoagulant activity of heparin is dependent on molecular weight and sulfate group. In recent years, non-anticoagulant activities such as anti-inflammation and anti-complement activation of heparin have attracted much attention. Alzheimer's disease (AD), also known as Alzheimer's disease (AD), is a degenerative disease of the nervous system that poses a great threat to the health of the elderly. P-amyloid (AD) deposits are thought to be a major component of age-related plaques and one of the pathogenesis of Alzheimer's disease. Ap deposits are thought to be caused by abnormal metabolism of amyloid precursor protein (APP). Oligomerized Ap and AP deposits can cause a series of brain damage. Neuritis, especially the activation of the complement system in the brain, is one of the important injury mechanisms of ap. It has been found that heparin plays an important role in APP metabolism-related enzymes and has an important impact on neuroinflammation caused by AD, but there is no systematic and in-depth study. The effects of heparin components with different molecular weights and sulfated degrees on APP metabolism and AD-induced complement activation were studied by using cell-like cells as cell models to elucidate the relationship between heparin structure and its anti-AD activity.
The research contents and achievements of this subject mainly include five aspects:
Construction of 1AD cell model
Neuron-like cell model was constructed by inducing differentiation of human neuroblastoma SH-SY5Y cells with RA. The morphology of SH-SY5Y cells was changed obviously after RA treatment, and the axon-like structure and cell markers were formed. The damage of neuron-like cells induced by A-beta 1-42 was determined by MTT colorimetric assay. Neuron like cells 24h can replicate the AD like cell model.
2 the survival rate of neuron like cells and AD like cells with different molecular weight and degree of sulfation.
The effects of heparin fractions with different molecular weight and sulfation degree on the survival rate of neuron-like cells and AD-like cells were determined by MTT colorimetry, including heparin, enoxaparin, 2-O-desulfated heparin, 6-O-desulfated heparin, N-desulfated-acetylated heparin, heparin disaccharide, heparin tetrasaccharide, heparin decase and heparin hexadecase. However, for AD-like cells, the molecular weight of AD-like cells was between 3000 and 5000 Da, and the components containing O-sulfate and N-sulfate groups, such as enoxaparin, heparin hexadecase and so on, could significantly improve the survival rate of AD-like cells and inhibit the damage of Ap to neuron-like cells.
3 the effect of different molecular weight and degree of sulfation on the metabolism of APP
Neuron-like cells injured by Ap were used as AD cell model for the first time to analyze the effects of Ap-1-42 on the key enzymes of APP metabolism, the related fragments of APP production and the effects of heparin and its derivatives on the above changes induced by Ap-1-42 in neuron-like cells. Enoxacin, heparin decase and heparin hexadecase had the strongest inhibitory effect on the formation of BACE-1. A beta 1-42 increased the expression of ADAM10 precursor and ADAM10, but not significantly. The results suggested that heparin sulfate, especially 6-O-sulfate and N-sulfate, and the molecular weight of sugars in the length of decase to hexadecase inhibited the production of AP-induced A. PP expression of metabolic key enzymes is essential.
A-beta-1-42 increased the expression of APP and decreased the expression of C99 in AD-like cells, but the expression of C83 remained unchanged. 2-O-desulfated heparin and heparin disaccharide, tetrasaccharide, decase and hexadecase increased the expression of C83 significantly, suggesting that heparin might have an important effect on the enzyme activity of ADAM10.
4 the effect of heparin components with different molecular weight and sulfation degree on complement system
Immunofluorescence showed that A-beta 1-42 promoted the expression of C5b-9 in neuron-like cells, and blocking CD59 did not affect the expression of C5b-9. Heparin could inhibit the expression of C5b-9 induced by A-beta 1-42.
The expression of proteins related to different systems in neuron-like cells was analyzed by Wlestem b10tting. The expression of C4a was significantly increased by A1-42. The inhibition of heparin and its derivatives on C4a expression was closely related to the sulfate group and molecular weight of heparin. O-sulfate and N-sulfate groups are necessary for heparin to inhibit C4a expression.
The expression of C3 was also significantly increased by A-beta 1-42, and the inhibitory effect of heparin and its derivatives was not significant.
These results suggest that heparin can inhibit neuron-like cell injury induced by Abeta by regulating APP metabolism and AP-induced complement activation, and this effect is related to the degree of sulfation of heparin, sulfate position and molecular weight. Ten heparin oligosaccharides with sugar to sixteen sugar and all sulfate groups, including O- sulfate and N- sulfate.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R749.16
[Abstract]:Heparin has been used only as anticoagulant and antithrombotic drugs in clinic. As a glycosaminoglycan, its structure is characterized by high molecular weight and high negative electric density. The anticoagulant activity of heparin is dependent on molecular weight and sulfate group. In recent years, non-anticoagulant activities such as anti-inflammation and anti-complement activation of heparin have attracted much attention. Alzheimer's disease (AD), also known as Alzheimer's disease (AD), is a degenerative disease of the nervous system that poses a great threat to the health of the elderly. P-amyloid (AD) deposits are thought to be a major component of age-related plaques and one of the pathogenesis of Alzheimer's disease. Ap deposits are thought to be caused by abnormal metabolism of amyloid precursor protein (APP). Oligomerized Ap and AP deposits can cause a series of brain damage. Neuritis, especially the activation of the complement system in the brain, is one of the important injury mechanisms of ap. It has been found that heparin plays an important role in APP metabolism-related enzymes and has an important impact on neuroinflammation caused by AD, but there is no systematic and in-depth study. The effects of heparin components with different molecular weights and sulfated degrees on APP metabolism and AD-induced complement activation were studied by using cell-like cells as cell models to elucidate the relationship between heparin structure and its anti-AD activity.
The research contents and achievements of this subject mainly include five aspects:
Construction of 1AD cell model
Neuron-like cell model was constructed by inducing differentiation of human neuroblastoma SH-SY5Y cells with RA. The morphology of SH-SY5Y cells was changed obviously after RA treatment, and the axon-like structure and cell markers were formed. The damage of neuron-like cells induced by A-beta 1-42 was determined by MTT colorimetric assay. Neuron like cells 24h can replicate the AD like cell model.
2 the survival rate of neuron like cells and AD like cells with different molecular weight and degree of sulfation.
The effects of heparin fractions with different molecular weight and sulfation degree on the survival rate of neuron-like cells and AD-like cells were determined by MTT colorimetry, including heparin, enoxaparin, 2-O-desulfated heparin, 6-O-desulfated heparin, N-desulfated-acetylated heparin, heparin disaccharide, heparin tetrasaccharide, heparin decase and heparin hexadecase. However, for AD-like cells, the molecular weight of AD-like cells was between 3000 and 5000 Da, and the components containing O-sulfate and N-sulfate groups, such as enoxaparin, heparin hexadecase and so on, could significantly improve the survival rate of AD-like cells and inhibit the damage of Ap to neuron-like cells.
3 the effect of different molecular weight and degree of sulfation on the metabolism of APP
Neuron-like cells injured by Ap were used as AD cell model for the first time to analyze the effects of Ap-1-42 on the key enzymes of APP metabolism, the related fragments of APP production and the effects of heparin and its derivatives on the above changes induced by Ap-1-42 in neuron-like cells. Enoxacin, heparin decase and heparin hexadecase had the strongest inhibitory effect on the formation of BACE-1. A beta 1-42 increased the expression of ADAM10 precursor and ADAM10, but not significantly. The results suggested that heparin sulfate, especially 6-O-sulfate and N-sulfate, and the molecular weight of sugars in the length of decase to hexadecase inhibited the production of AP-induced A. PP expression of metabolic key enzymes is essential.
A-beta-1-42 increased the expression of APP and decreased the expression of C99 in AD-like cells, but the expression of C83 remained unchanged. 2-O-desulfated heparin and heparin disaccharide, tetrasaccharide, decase and hexadecase increased the expression of C83 significantly, suggesting that heparin might have an important effect on the enzyme activity of ADAM10.
4 the effect of heparin components with different molecular weight and sulfation degree on complement system
Immunofluorescence showed that A-beta 1-42 promoted the expression of C5b-9 in neuron-like cells, and blocking CD59 did not affect the expression of C5b-9. Heparin could inhibit the expression of C5b-9 induced by A-beta 1-42.
The expression of proteins related to different systems in neuron-like cells was analyzed by Wlestem b10tting. The expression of C4a was significantly increased by A1-42. The inhibition of heparin and its derivatives on C4a expression was closely related to the sulfate group and molecular weight of heparin. O-sulfate and N-sulfate groups are necessary for heparin to inhibit C4a expression.
The expression of C3 was also significantly increased by A-beta 1-42, and the inhibitory effect of heparin and its derivatives was not significant.
These results suggest that heparin can inhibit neuron-like cell injury induced by Abeta by regulating APP metabolism and AP-induced complement activation, and this effect is related to the degree of sulfation of heparin, sulfate position and molecular weight. Ten heparin oligosaccharides with sugar to sixteen sugar and all sulfate groups, including O- sulfate and N- sulfate.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R749.16
【共引文献】
相关期刊论文 前10条
1 胡莉琴;王春芝;唐震宇;;N-乙酰天门冬氨酸在神经变性病中的诊断价值[J];广东医学;2013年19期
2 饶艳秋;王文君;;雌激素防治阿尔茨海默病的作用机制[J];国际妇产科学杂志;2014年01期
3 李鑫;黄晏;胡增\，
本文编号：2242484
本文链接：https://www.wllwen.com/yixuelunwen/jsb/2242484.html
最近更新
教材专著
