本文關(guān)鍵詞：褐藻膠及其衍生物的免疫抗炎及抗阿爾茲海默癥的機(jī)制研究　出處：《深圳大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 褐藻膠 寡糖 硒化甘露糖醛酸 吞噬作用 抗炎 自噬 阿爾茲海默癥

【摘要】：褐藻膠是由互為C5差向異構(gòu)體的聚甘露糖醛酸(PM)和聚古羅糖醛酸(PG)相互嵌合而成的線性大分子,是一類廣泛存在于各種褐藻中的酸性多糖。據(jù)大量文獻(xiàn)報(bào)道,褐藻膠及其衍生物具有多種生物活性。本課題以褐藻膠、硒化甘露糖醛酸(Se-PM)、酶解甘露糖醛酸寡糖(MOS)為原料,分別研究它們的促吞噬作用、抗炎、抗阿爾茲海默癥(AD)等方面的生物活性。有以下四個(gè)工作:(1)本課題研究褐藻膠促進(jìn)小鼠巨噬細(xì)胞RAW264.7吞噬作用的機(jī)制。研究發(fā)現(xiàn),褐藻膠能夠顯著提高RAW264.7細(xì)胞對(duì)金納米顆粒、熒光微球、免疫球蛋白G(IgG)調(diào)理的金黃色葡萄球菌的吞噬作用。究其機(jī)制,形態(tài)學(xué)分析表明褐藻膠能夠明顯提高RAW264.7細(xì)胞面積。此外,深入研究發(fā)現(xiàn)褐藻膠能夠顯著上調(diào)RAW264.7細(xì)胞中Toll樣受體4(TLR4)基因和蛋白的表達(dá),當(dāng)用TLR4的特異性抑制劑TAK-242處理細(xì)胞及利用shRNA干擾技術(shù)將RAW264.7細(xì)胞的TLR4基因下調(diào)后,褐藻膠促進(jìn)RAW264.7細(xì)胞吞噬金納米顆粒的作用被顯著性抑制。研究還發(fā)現(xiàn),褐藻膠能夠激活RAW264.7細(xì)胞核轉(zhuǎn)錄因子κB(NF-κB)信號(hào)通路和p38絲裂原活化蛋白激酶(MAPK)信號(hào)通路,當(dāng)用p38 MAPK和NF-κB的抑制劑處理細(xì)胞后,褐藻膠促進(jìn)巨噬細(xì)胞吞噬IgG調(diào)理的金黃色葡萄球菌的作用有明顯的降低。(2)本課題研究Se-PM的抗炎活性及分子機(jī)制。研究發(fā)現(xiàn),Se-PM能夠有效抑制LPS誘導(dǎo)的小鼠巨噬細(xì)胞RAW264.7細(xì)胞炎癥介質(zhì),如一氧化氮(NO)、前列腺素E2(PGE2)、活性氧簇(ROS)的生成,誘導(dǎo)型一氧化氮合酶(iNOS)、環(huán)氧合酶-2(COX-2)的表達(dá),細(xì)胞因子,如腫瘤壞死因子α(TNF-α)、白介素1β(IL-1β)、IL-6、IL-12的分泌,且均呈濃度依賴性。深入研究發(fā)現(xiàn),Se-PM可以顯著抑制LPS誘導(dǎo)的RAW264.7細(xì)胞NF-κB信號(hào)通路和MAPK信號(hào)通路的過(guò)度激活。并且,Se-PM還能夠顯著抑制LPS誘導(dǎo)的原代小鼠巨噬細(xì)胞炎癥介質(zhì),如NO、TNF-α、IL-1β和IL-6的生成。另外,本課題構(gòu)建LPS誘導(dǎo)的敗血性休克小鼠模型和卡拉膠誘導(dǎo)的氣囊炎癥小鼠模型,在動(dòng)物水平上研究發(fā)現(xiàn),Se-PM能夠抑制上述兩種炎癥模型的小鼠腹水、血清、氣囊浸出液中TNF-α和IL-6的分泌。(3)本課題研究MOS通過(guò)自噬途徑降低AD模型細(xì)胞的Aβ生成。研究發(fā)現(xiàn),MOS可以降低N2a-sw-APP695細(xì)胞中β-淀粉樣蛋白(Aβ)的分泌、淀粉樣前體蛋白(APP)和β分泌酶(BACE1)的表達(dá),抑制3×Transgene(3×Tg,APPswe/PS1M146V/TauP301L)小鼠原代神經(jīng)元細(xì)胞中APP和BACE1的表達(dá)。并且,MOS還可以降低哺乳動(dòng)物雷帕霉素靶蛋白(mTOR)的激活,提高Beclin-1、微管相關(guān)蛋白輕鏈3(LC3)-II、組織蛋白酶(Cat)-D的表達(dá),抑制p62的表達(dá),進(jìn)而提高N2a-sw-APP695細(xì)胞的自噬水平。(4)本課題研究MOS通過(guò)自噬途徑降低Tau蛋白磷酸化。研究發(fā)現(xiàn),MOS可以降低HEK293/Tau細(xì)胞和3×Tg小鼠原代神經(jīng)元細(xì)胞中Tau蛋白在絲氨酸202、262、396、404位點(diǎn)的磷酸化水平,以及總Tau的表達(dá)水平,還可以抑制糖原合成酶激酶3β(GSK3β)的活性。深入研究還發(fā)現(xiàn),MOS可以降低3×Tg小鼠原代神經(jīng)元細(xì)胞mTOR的激活,提高Beclin-1、LC3-II、Cat-D的表達(dá),降低p62的表達(dá),進(jìn)而提高細(xì)胞自噬,促進(jìn)自噬體和溶酶體的融合。本論文發(fā)現(xiàn)褐藻膠及其衍生物在促吞噬作用、抗炎、抗AD方面具有良好的生物活性,具有開發(fā)成各類保健食品或藥物的潛力。
[Abstract]:Alginate is by mutual C5 epimer of Polymannuronic acid (PM) and poly guluronate (PG) linear macromolecules mutually embedded together, is a kind of acidic polysaccharide alginate in widely exists in various. According to a large number of reports, the alginate and its derivatives have a variety of bioactivity. In this study, alginate, seleno manuronic acid (Se-PM) and mannan uronic acid oligosaccharides (MOS) were used as raw materials to study their phagocytosis, anti inflammation and anti Alzheimer's disease (AD), respectively. There are four tasks as follows: (1) the mechanism of the phagocytosis of murine macrophage RAW264.7 by algin is studied in this study. It is found that alginate can significantly enhance the phagocytosis of RAW264.7 cells on Staphylococcus aureus, which is modulated by gold nanoparticles, fluorescent microspheres and immunoglobulin G (IgG). The morphological analysis showed that brown alga could significantly increase the area of RAW264.7 cells. In addition, we found that alginate can significantly increase RAW264.7 cell Toll like receptor 4 (TLR4) gene and protein expression, when using the TLR4 and the cells treated with TAK-242 specific inhibitor shRNA interference TLR4 gene of RAW264.7 cells decreased after alginate promotes RAW264.7 cell phagocytosis of gold nanoparticles is significant suppression. The study also found that alginate can activate RAW264.7 nuclear transcription factor kappa B (NF- K B) signaling pathway and p38 mitogen activated protein kinase (MAPK) signaling pathway, when using the p38 MAPK and NF- kappa B inhibitor treatment cells, alginate IgG promote macrophage phagocytosis of opsonized Staphylococcus aureus have obvious effect reduce. (2) this topic studies the anti-inflammatory activity and molecular mechanism of Se-PM. The study found that Se-PM can effectively inhibit the mouse macrophage RAW264.7 cells LPS induced by inflammatory mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), reactive oxygen species (ROS) generation of inducible nitric oxide synthase (iNOS) and cyclooxygenase -2 (COX-2) on the surface of cells, such as tumor necrosis factor. Factor alpha (TNF- alpha), interleukin 1 beta (IL-1 beta), the secretion of IL-6 and IL-12, and were in a concentration dependent manner. It was found that Se-PM could significantly inhibit the overactivation of NF- kappa B signaling pathway and MAPK signaling pathway in RAW264.7 cells induced by LPS. Moreover, Se-PM can also significantly inhibit the LPS induced macrophage inflammatory mediators, such as the formation of NO, TNF- alpha, IL-1 beta and IL-6. In addition, the construction of airbag inflammation in mice model of septic shock mice model and carrageenan induced by LPS induction, the study found in the animal level, secretion of TNF- and IL-6 Se-PM can inhibit the two inflammatory model mice ascites, serum, balloon in leaching solution. (3) the study of MOS reduces the A beta production of AD model cells by autophagy pathway. It is found that MOS can reduce the expression of beta amyloid protein (A beta), the expression of amyloid precursor protein (APP) and beta secretase (BACE1) in N2a-sw-APP695 cells, and inhibit the expression of APP and BACE1 in primary neurons of 3 * Transgene (3 x Tg, APPswe/PS1M146V/TauP301L) mice. Moreover, MOS can also reduce the activation of mammalian target of rapamycin (mTOR), increase the expression of Beclin-1, microtubule associated protein light chain 3 (LC3) -II and cathepsin (Cat) -D, inhibit the expression of p62, and further improve the autophagy level of N2a-sw-APP695 cells. (4) this topic studies MOS to reduce the phosphorylation of Tau protein by autophagy pathway. It is found that MOS can reduce the phosphorylation level of Tau protein at serine 202, 262, 396, 404 sites, and the expression level of total Tau in HEK293/Tau cells and 3 * Tg mouse primary neurons, and also inhibit the activity of glycogen synthase kinase 3 GSK3 (GSK3 beta). Further studies also showed that MOS could reduce the activation of mTOR in primary neurons of 3 * Tg mice, increase the expression of Beclin-1, LC3-II and Cat-D, reduce the expression of p62, further enhance autophagy, and promote the fusion of autophagosome and lysosome. It is found that alginate and its derivatives have good bioactivity in promoting phagocytosis, anti-inflammatory and anti AD, and have potential to develop various kinds of health foods or drugs.
【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285

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