【摘要】：肝素一直以來僅作為抗凝和抗血栓藥物用于臨床,作為一種糖胺聚糖,其結(jié)構(gòu)特點(diǎn)是高分子和高負(fù)電密度；從已經(jīng)闡明的抗凝活性看,其活性是分子量和硫酸基所依賴的。近年來,肝素的抗炎、抗補(bǔ)體激活等非抗凝活性也得到關(guān)注,但尚未得到應(yīng)用。阿爾茨海默病(Alzheimer's disease, AD),又稱早老性癡呆癥,是一種神經(jīng)系統(tǒng)退行性疾病,對(duì)老年人的健康產(chǎn)生極大威脅。AD的特征性病理學(xué)改變?yōu)榇竽X皮質(zhì)神經(jīng)細(xì)胞內(nèi)神經(jīng)原纖維纏結(jié)、細(xì)胞外大量老年斑形成和大腦皮質(zhì)細(xì)胞減少。p-淀粉樣蛋白(β-amyloid, AD)沉積被認(rèn)為是老年斑的主要成分,也是阿爾茨海默病的發(fā)病機(jī)制之一。Ap的沉積被認(rèn)為是由淀粉樣前體蛋白(amyloid precursor protein, APP)的代謝異常導(dǎo)致的。寡聚化的Ap和Ap沉積物又會(huì)引發(fā)對(duì)腦組織的一系列損傷,最終導(dǎo)致AD病變。其中,神經(jīng)炎癥,特別是對(duì)腦內(nèi)補(bǔ)體系統(tǒng)的激活,是Ap的重要損傷機(jī)制之一。已有研究發(fā)現(xiàn),肝素對(duì)APP代謝相關(guān)酶有著重要作用,對(duì)AD引起的神經(jīng)炎癥也存在著重要影響,但缺乏系統(tǒng)和深入的研究。本課題利用Ap損傷的神經(jīng)元樣細(xì)胞作為細(xì)胞模型,研究不同分子量和硫酸化程度肝素組分對(duì)APP代謝和AD引發(fā)的補(bǔ)體激活的作用,以闡明肝素結(jié)構(gòu)與其抗AD活性的關(guān)系。 本課題的研究?jī)?nèi)容及成果主要包括五個(gè)方面： 1AD細(xì)胞模型的構(gòu)建 通過RA誘導(dǎo)分化人神經(jīng)母細(xì)胞瘤細(xì)胞SH-SY5Y構(gòu)建神經(jīng)元樣細(xì)胞模型。RA處理使SH-SY5Y的形態(tài)發(fā)生明顯變化,形成神經(jīng)元細(xì)胞特有的軸突的結(jié)構(gòu)和細(xì)胞標(biāo)記。利用MTT比色法測(cè)定Aβ1-42對(duì)神經(jīng)元樣細(xì)胞的損傷。確定用濃度為100μg/mL的Aβ1-42處理神經(jīng)元樣細(xì)胞24h可復(fù)制AD樣細(xì)胞模型。 2不同分子量和硫酸化程度肝素組分對(duì)神經(jīng)元樣細(xì)胞和AD樣細(xì)胞存活率的研究 利用MTT比色法測(cè)定了不同分子量和硫酸化程度的肝素組分對(duì)神經(jīng)元樣細(xì)胞和AD樣細(xì)胞存活率的影響,包括肝素、依諾肝素、2-O-脫硫酸肝素、6-O-脫硫酸肝素、N-脫硫酸-乙�；嗡�、肝素二糖、肝素四糖、肝素十糖和肝素十六糖。不同肝素組分對(duì)神經(jīng)元樣細(xì)胞存活率沒有顯著性影響。而對(duì)于AD樣細(xì)胞,分子量在3000-5000Da,且含有O-硫酸基和N-硫酸基的肝素組分,如依諾肝素、肝素十六糖等,能顯著提高AD樣細(xì)胞的存活率、抑制Ap對(duì)神經(jīng)元樣細(xì)胞的損傷的活性在所有肝素組分中。 3不同分子量和硫酸化程度肝素組分對(duì)APP代謝作用的研究 首次利用Ap損傷的神經(jīng)元樣細(xì)胞作為AD細(xì)胞模型,分析了Aβ1-42對(duì)神經(jīng)元樣細(xì)胞的APP代謝關(guān)鍵酶、APP和生成的相關(guān)片段的影響,以及肝素及其衍生物對(duì)Aβ1-42引起的上述變化的影響。Aβ1-42顯著提高了BACE1的表達(dá)量,而肝素及其衍生物對(duì)其具有抑制作用,依諾肝素、肝素十糖和肝素十六糖對(duì)BACE-1生成的抑制作用最強(qiáng)。Aβ1-42提高了ADAM10前體及ADAM10表達(dá)量,但并不顯著。結(jié)果提示肝素的硫酸基,特別是6-O-硫酸基和N-硫酸基,分子量在十糖到十六糖長(zhǎng)度的糖鏈對(duì)肝素抑制Ap引起的APP代謝關(guān)鍵酶表達(dá)是必需的。 Aβ1-42提高了AD樣細(xì)胞的APP表達(dá)量,降低了C99的表達(dá)量,而C83的表達(dá)量基本不變,但這些影響并不顯著。2-O-脫硫酸肝素和肝素二糖、四糖、十糖、十六糖使C83表達(dá)量明顯增加,提示肝素對(duì)ADAM10的酶活力可能存在著重要影響。 4不同分子量和硫酸化程度肝素組分對(duì)補(bǔ)體系統(tǒng)作用的研究 免疫熒光檢查發(fā)現(xiàn),Aβ1-42促進(jìn)神經(jīng)元樣細(xì)胞表達(dá)C5b-9,而且封閉CD59對(duì)表達(dá)量并無(wú)明顯影響。肝素能抑制Aβ1-42引起的C5b-9表達(dá)。 首次利用Aβ1-42損傷的神經(jīng)元樣細(xì)胞作為AD細(xì)胞模型,通過Wlestem b10tting分析了神經(jīng)元樣細(xì)胞細(xì)胞中不同系統(tǒng)相關(guān)蛋白的表達(dá)情況。Aβ1-42顯著促進(jìn)了C4a的表達(dá),而肝素及其衍生物對(duì)C4a表達(dá)的抑制作用與肝素的硫酸基及分子量密切相關(guān)。6-O-硫酸基和N-硫酸基對(duì)于肝素抑制C4a表達(dá)的活性是必需的。十六糖或以上的肝素糖鏈才可能具有抑制C4a表達(dá)的活性。 Aβ1-42也能顯著提高C3的表達(dá)量,肝素及其衍生物對(duì)其的抑制作用不具有顯著性影響。 以上結(jié)果表明,肝素可通過調(diào)節(jié)APP代謝和Ap引起的補(bǔ)體系統(tǒng)激活抑制Aβ引起的對(duì)神經(jīng)元樣細(xì)胞的損傷,且這種作用都與肝素的硫酸化程度、硫酸基位置和分子量有關(guān)。從調(diào)節(jié)APP代謝和抑制補(bǔ)體激活的角度來看,具有抗AD活性的肝素應(yīng)為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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R749.16

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 胡莉琴;王春芝;唐震宇;;N-乙酰天門冬氨酸在神經(jīng)變性病中的診斷價(jià)值[J];廣東醫(yī)學(xué);2013年19期

2 饒艷秋;王文君;;雌激素防治阿爾茨海默病的作用機(jī)制[J];國(guó)際婦產(chǎn)科學(xué)雜志;2014年01期

3 李鑫;黃晏;胡增\，

本文編號(hào)：2242484