本文關(guān)鍵詞： 老年癡呆癥 米糠黃酮 提取 銅離子 β-淀粉樣多肽　出處：《中南林業(yè)科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：老年癡呆癥是常見的神經(jīng)退行性疾病,患者具有大腦萎縮、細(xì)胞外出現(xiàn)淀粉樣斑塊、細(xì)胞內(nèi)神經(jīng)纖維纏結(jié)、神經(jīng)元大量喪失,以及金屬離子的過量堆積等臨床病理特征。近年來的研究表明:金屬離子(如Cu2+)引起的氧化應(yīng)激和異常聚集的Aβ可能是導(dǎo)致AD的主要原因。具有氧化還原活性的Cu2+能催化產(chǎn)生自由基,破壞線粒體的正常生理功能,誘導(dǎo)細(xì)胞膜、組織和酶氧化損傷,最終導(dǎo)致神經(jīng)變性疾病;Aβ聚集形成的聚集物(寡聚體、纖維等)可以破壞細(xì)胞膜,引起炎癥反應(yīng),導(dǎo)致神經(jīng)元凋亡。本文以米糠為原料,提取其中的粗黃酮,研究米糠粗黃酮對金屬離子引起的氧化應(yīng)激和異常聚集的Aβ導(dǎo)致的神經(jīng)毒性的影響。利用CCA熒光法、原子力顯微鏡、噻哇藍(lán)、MTT實(shí)驗(yàn)、Hoechst 33258染色實(shí)驗(yàn)、DCFH-DA測內(nèi)源性RO實(shí)驗(yàn)、western blot實(shí)驗(yàn)探討了米糠粗黃酮(Crude flavonoids from Rice bran,FRB)抑制羥基自由基和 Aβ誘導(dǎo)的 SH-SY5Y細(xì)胞凋亡的作用及機(jī)理的初步研究。通過本論文的研究發(fā)現(xiàn):1.利用酶解輔助超聲的方法提取米糠粗黃酮并對其提取工藝進(jìn)行優(yōu)化,米糠粗黃酮的提取率達(dá)到3.26%。HPLC初步檢測米糠粗黃酮中含有柚皮素成分,含量占4.47%。2.米糠粗黃酮能減少Cu2+催化H2O2、Cu2+誘導(dǎo)抗壞血酸(AA)產(chǎn)生的羥基自由基。CCA熒光實(shí)驗(yàn)表明米糠粗黃酮能分別使Cu2+催化H202、Cu2+誘導(dǎo)AA產(chǎn)生的羥基自由基的量降低61.82%、57.46%。通過MTT實(shí)驗(yàn)發(fā)現(xiàn)60 μg/mL的米糠粗黃酮使羥基自由基損傷的SH-SY5Y細(xì)胞的存活率由(25.97±2.06)%增加至(76.96±4.39)%。因此,米糠粗黃酮可以通過抑制羥基自由基的生成,從而抑制SH-SY5Y細(xì)胞的凋亡。3.米糠粗黃酮能抑制Aβ(1-42)毒性聚集物的產(chǎn)生。利用AFM檢測發(fā)現(xiàn)米糠粗黃酮能減少Aβ(1-42)毒性聚集物(寡聚體、纖維前體、纖維)的產(chǎn)生,改變Aβ(1-42)的聚集路徑。在一定濃度范圍內(nèi),米糠粗黃酮濃度越高,抑制Aβ(1-42)聚集物生成的效果更明顯。4.米糠粗黃酮能抑制Aβ(1-42)毒性聚集物誘導(dǎo)的SH-SY5Y細(xì)胞的凋亡。通過MTT和Hoechst 33258實(shí)驗(yàn)發(fā)現(xiàn)80μM Aβ(1-42)處理的細(xì)胞存活率為(52.25±1.34)%,且細(xì)胞呈極亮的藍(lán)色。120μM米糠粗黃酮使SH-SY5Y細(xì)胞的存活率由(52.25±1.34)%增加至(91.47±1.45)%,細(xì)胞極亮的藍(lán)光減少,細(xì)胞核形態(tài)呈均一飽滿狀。說明米糠粗黃酮能明顯抑制Aβ(1-42)誘導(dǎo)的SH-SY5Y細(xì)胞的凋亡。5.米糠粗黃酮能減少細(xì)胞內(nèi)源性ROS和下調(diào)caspase-3,從而抑制Aβ(l-42)誘導(dǎo)SH-SYSY細(xì)胞的凋亡。通過DCFH-DA測細(xì)胞內(nèi)源性ROS發(fā)現(xiàn),米糠粗黃酮使細(xì)胞內(nèi)源性ROS由1.72±0.02降低到1.12±0.04,從而保護(hù)SH-SY5Y細(xì)胞免受Aβ(1-42)誘導(dǎo)的損傷。western blot實(shí)驗(yàn)結(jié)果表明Aβ(1-42)能極顯著性(p0.01)的上調(diào)細(xì)胞caspase-3的表達(dá),而120 μM的米糠粗黃酮使caspase-3的蛋白表達(dá)相對量由1.73± 0.07降低到1.07±0.02。因此米糠粗黃酮可以通過下調(diào)caspase-3,從而抑制Aβ(1-42)誘導(dǎo)的SH-SY5Y細(xì)胞凋亡。綜上,本文對米糠粗黃酮的提取工藝進(jìn)行優(yōu)化,并利用HPLC初步測定其成分;研究了米糠粗黃酮對羥基自由基和Aβ誘導(dǎo)SH-SY5Y凋亡的抑制作用,并對其機(jī)理進(jìn)行了初步探究;為米糠的綜合利用提供了新的研究方向,為AD的治療提供了新思路,為開發(fā)新的治療藥物提供了理論依據(jù),具有重大的現(xiàn)實(shí)意義。
[Abstract]:Alzheimer's disease is a common neurodegenerative disease, patients with brain atrophy, extracellular amyloid plaques appear, intracellular neurofibrillary tangles, neuronal loss, excessive accumulation of metal ions and the clinical pathological features. Recent studies show that metal ions (such as Cu2+) induced by oxidative stress and abnormal aggregation A beta may be the main cause of AD. Cu2+ has redox activity to catalyze the production of free radicals, the normal physiological function of mitochondria induced damage, cell membrane, tissue and enzymatic oxidative damage, eventually lead to neurodegenerative diseases; the aggregates formed by the aggregation of beta A (oligomers, fibers) can destroy the cell membrane, causing inflammation leads to neuronal apoptosis. In this paper, rice bran as raw material, the extraction of crude flavonoids from oxidative stress and the differentiation of rice bran flavonoids due to metal ions used to gather A caused by God The toxicity effect. By using CCA fluorescence spectrometry, atomic force microscopy, thiophene wow blue, MTT assay, Hoechst 33258 staining experiments, measurement of endogenous RO DCFH-DA Western blot experiment, experimental study of rice bran flavonoids (Crude flavonoids from Rice bran, FRB) a preliminary study on the inhibitory effect and mechanism of apoptosis of SH-SY5Y cells and A beta hydroxyl radicals the induction. Through the research of this paper found: 1. using enzyme assisted ultrasonic method for the extraction of rice bran crude flavonoids and the extraction process was optimized, the extraction rate of rice bran crude flavonoids to contain naringin component 3.26%.HPLC preliminary detection of yellow ketone content in rice bran, rice bran flavonoids can reduce 4.47%.2. catalyzed by Cu2+ H2O2. Cu2+ induced by ascorbic acid (AA) produced by hydroxyl radical.CCA fluorescence experiments showed that rice bran crude flavonoids respectively catalyzed by Cu2+ H202, Cu2+ AA induced by hydroxyl radicals generated was reduced by 61.82%, 57.46%. Through the MTT experiment found that rice bran flavonoids of 60 g/mL hydroxyl free radical damage to the survival rate of SH-SY5Y cells by (25.97 + 2.06)% increased to (76.96 + 4.39)%. Therefore, the rice bran flavonoids by inhibiting the production of hydroxyl radicals, the apoptosis of.3. rice bran flavonoids inhibit SH-SY5Y cell can inhibit A beta (1-42) toxic aggregates. Found that rice bran crude flavonoids can reduce A beta detected by AFM (1-42) toxic aggregates (oligomers, fiber precursor fiber) generation, change A beta (1-42) aggregation path. In a certain range of concentration, the higher the concentration of Flavonoids from rice bran. Inhibition of A beta (1-42) aggregates generated more obvious effect of.4. rice bran crude flavonoids can inhibit the apoptosis of A beta (1-42) toxic aggregates of SH-SY5Y cells induced by MTT and Hoechst. 33258 experiments showed that 80 M A beta (1-42) the cell survival rate was (52.25 + 1.34)%, and the cells a very bright blue.1 20 M rice bran flavonoids increased the survival rate of SH-SY5Y cells by (52.25 + 1.34)% increased to (91.47 + 1.45)%, bright blue cells decreased, nuclear morphology showed uniform full shape. The rice bran crude flavonoids can inhibit the apoptosis of.5. A beta (1-42) rice bran flavonoids induced SH-SY5Y cell to reduce the endogenous ROS and down-regulation of Caspase-3, thereby inhibiting A beta (L-42) induced apoptosis in SH-SYSY cells. DCFH-DA cells by measuring endogenous ROS found that rice bran flavonoids increased endogenous ROS from 1.72 + 0.02 reduced to 1.12 + 0.04, which protects SH-SY5Y cells against A beta (1-42) injury induced by.Western blot experiments show that A beta (1-42) can significantly (P0.01) expression of Caspase-3 cells, and rice bran crude flavonoid 120 M caspase-3 protein relative amount from 1.73 + 0.07 to 1.07 + 0.02. therefore reduce rice bran crude flavonoids by down regulating the expression of caspase-3, Thus inhibition of A beta (1-42) induced apoptosis in SH-SY5Y cells. In conclusion, this paper optimize the extraction process of rice bran crude flavonoids, and preliminary determination of its composition by HPLC; study the inhibitory effect of rice bran flavonoids on hydroxyl radical and A beta induced SH-SY5Y apoptosis, and the mechanism provides a preliminary inquiry; a new research direction for comprehensive utilization of rice bran, provides a new idea for the treatment of AD, and provides a theoretical basis for the development of new therapeutic agents, is of great practical significance.

【學(xué)位授予單位】：中南林業(yè)科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R151.2
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本文編號：1455203