【作者】 冯颖；

【导师】 孙桂媛； 刘瑞田；

【作者基本信息】 中国医科大学 ， 人体解剖与组织胚胎学， 2010， 博士

【摘要】 目的阿尔茨海默病(Alzheimer’s Disease, AD)是以记忆力减退、认知功能障碍为特征的中枢神经系统变性疾病,病情呈进行性加重,在几年内丧失独立生活能力,10年左右常因并发感染而死亡。由于老年人群的增长,AD已成为现代社会的常见病,引起了研究者们的关注。AD的典型病理特征是神经细胞内出现神经元纤维缠结,神经细胞外存在神经炎斑或称老年斑,老年斑的中心为β淀粉样蛋白(β-amyloid, Aβ)。Aβ是含有40-42个氨基酸的多肽,可诱导神经元的凋亡和突触丢失,损伤胆碱能神经元,影响细胞Ca2+的平衡,Aβ诱导产生的活性氧自由基可破坏细胞膜,促使脂质过氧化和膜蛋白损伤。生理情况下Ap是可溶的,低浓度不表现神经毒性,当可溶性的Aβ达到了临界浓度,就会发生构象的变化,从单体聚集形成具有稳定构象的寡聚体或纤维。Aβ在AD病人脑中有几种不同形式,包括可溶性的单体、寡聚体和不可溶的纤维。其中,寡聚体是毒性最大的。Aβ具有很强的自我聚集能力,寻找一些分子阻止或干扰Ap聚集或减轻其毒性已成为AD治疗的理想靶标。研究表明,适当的饮用红葡萄酒可以减轻AD病人的认知功能障碍及淀粉样沉积的病理变化。红葡萄酒中含有大量的多酚类物质,这些多酚类物质可以与一些肽或蛋白结合,起到了预防和治疗不同疾病的作用。多酚类物质具有潜在的抗淀粉样蛋白活性,从葡萄籽中提取出来的多酚类物质可以减少Aβ产生,抑制Aβ聚集和毒性,姜黄素也能有效减少脑内Aβ毒性和小胶质细胞的激活。本研究以毒性较大的Aβ42为研究对象,探讨了两种多酚类物质白藜芦醇(Resveratrol, Res)和鞣花酸(Ellagic acid, EA)对其聚集和细胞毒性的影响,为寻找预防和治疗AD的理想药物提供依据。另外,在实验中通过比较Aβ氨基端多肽的细胞毒性,发现高浓度Aβ1-16具有明显的细胞毒性,且该毒性作用呈剂量依赖关系,因此针对Aβ1-16的毒性及其是否引发炎症反应进行了一系列的研究。实验方法1、Res对Aβ42聚集及细胞毒性的影响(1)将不同浓度Res(0、2、1 0、1 00μM)与Aβ42(10μM)37℃共同孵育,分别于0 h、7 h、14 h、21 h、28 h取样,通过硫磺素T(Thioflavin T,ThT)荧光方法检测Res对Aβ42聚集的影响；(2)Aβ42(10μM)与Res(0、10、1 00μM)共同孵育,分别于28 h和3 d各取样1 0μL,通过透射电镜(Transmission Electron Microscopy, TEM)观察Res对Aβ42聚集过程的影响；(3)Aβ42(40μM)与Res(400μM)共同孵育,以单独孵育的Aβ42为对照,分别于0 h、11 h、15 h、18 h、24 h取样,通过圆二色谱仪(Circular Dichroism,CD)检测Res对Aβ42二级结构变化的影响；(4)酶联免疫吸附试验(Enzyme-linked immunosorbent assay, Elisa)和斑点印迹法(dot-blot)检测加入Res后,不同时间点Aβ42寡聚体含量的改变；(5)western-blot观察Res对Aβ42聚集的影响和对Aβ42纤维的解聚作用；(6)细胞毒性分析实验(MTT法)检测Res对Aβ42所致细胞毒性的影响。2、EA对Aβ42聚集及细胞毒性的影响(1) Aβ42 (10μM)和EA(100μM)共同孵育,分别于2 h、6 h、12 h、24 h取样10μL,以单独孵育的Aβ42为对照,观察聚集过程的变化；(2)Aβ42与EA共同孵育6 h,通过western-blot方法检测与Aβ42单独孵育形成蛋白的差异；(3) Aβ42 (10μM)和EA (100μM、300μM)共同孵育1 2 h和24 h,以单独孵育的Aβ42为对照,Elisa方法检测EA对Aβ42聚集形成寡聚体含量的影响；(4) Aβ42 (40μM)与EA (300μM)共同孵育,分别于0 h、4 h、6 h取样,以单独孵育的Aβ42为对照,通过圆二色潜仪检测EA对Aβ42二级结构变化的影响；(5)MTT法检测不同浓度EA对Aβ42所致细胞毒性的影响。3、Aβ1-16毒性及炎症反应的研究(1)MTT法比较Aβ42 N端多肽的细胞毒性；(2)MTT法测定不同浓度Aβ1-16的细胞毒性；(3)不同孵育温度、时间对Aβ1-16细胞毒性的影响；(4)利用Elisa试剂盒检测Aβ1-16对BV-2小胶质细胞释放炎症因子的影响；(5)小鼠脑海马区定位注射Aβ1-16；(6)利用Morris水迷宫测定小鼠的学习和空间记忆能力；(7)免疫组化检测注射Aβ1-16后,星形胶质细胞的表达变化。结果1、Res对Aβ42聚集及细胞毒性的影响(1)ThT、TEM和CD的结果表明,Res抑制Aβ42的聚集；(2) Elisa、Dot-blo、western-blot和TEM结果表明,Res不抑制Aβ42寡聚体的形成,稳定寡聚体的存在；(3)ThT, western-blot和TEM结果表明,Res对Aβ42纤维具有解聚作用；(4)MTT结果表明,与2μM Res共同孵育使Aβ42对SH-SY5Y细胞的毒性明显降低,10μMRes使Aβ42纤维所致的细胞毒性明显降低。2、EA对Aβ42聚集及细胞毒性的影响(1)TEM和western-blot结果表明,EA加速Aβ42的聚集；(2)Elisa结果表明,与EA共同孵育12 h和24 h后,显著减少了Aβ42寡聚体的含量；(3)CD结果表明,加入EA后,在215 nm形成更大的负峰,说明EA加速了β-片层结构的形成；(4)MTT结果表明,EA显著降低了Aβ42引起的细胞毒性。3、Aβ1-16毒性及炎症反应的研究(1)Aβ1-1 6以30μM作用于SH-SY5Y细胞48 h后,细胞数量明显减少,形态不规则,边缘不清晰,有聚堆现象；(2)MTT结果表明,Aβ1-16对SH-SY5Y细胞的毒性呈剂量依赖的形式,随着浓度的增加,细胞毒性逐渐增加；(3)将Aβ1-16分别于4℃,37℃孵育3 d和7 d,结果表明,随着孵育时间的延长,Aβ1-16引起的细胞毒性逐渐下降；(4)Aβ1-16(30、100μM)刺激了BV-2小胶质细胞TNF-α的分泌,不影响IL-1β的分泌；100μM的Aβ1-16明显提高了BV-2小胶质细胞分泌IL-4的水平；(5)水迷宫实验结果：与对照组和Aβ1-8组相比,Aβ1-16组的学习和空间记忆能力要差。(6)免疫组化结果表明,脑海马区注射Aβ1-16后,引起了星形胶质细胞的激活。结论1、Res抑制Aβ42聚集成纤维,但不抑制寡聚体的形成；2、Res对Aβ42纤维具有解聚作用；3、Res可以减轻Aβ42对SH-SY5Y细胞的毒性；4、EA促进Aβ42聚集,减少寡聚体存在的时间,并抑制Aβ42所致的细胞毒性；5、Res和EA影响Aβ42聚集的路径不同,但都减轻了Aβ42的细胞毒性,一方面可能由于它们的抗氧化作用,另一方面则由于它们与Aβ42结合后,可能改变了毒性较大的寡聚体的构象,从而减轻了细胞毒性；6、Aβ1-16具有细胞毒性,且呈浓度依赖关系；并能刺激BV-2小胶质细胞炎症因子TNF-α及IL-4的分泌；海马区注射Aβ1-16后,会导致记忆损伤,增加星形胶质细胞的表达。更多还原

【Abstract】 ObjectiveAlzheimer’s disease (AD) is a neurodegenerative disorder with insidious onset and progression of cognitive decline. AD has achieved more and more researchers’ attention because of the increasing of old people. Histopathologically, AD is characterized by extracellular "senile plaques" containing amyloidβ-peptide (AP) fibrils and intracellular "neurofibrillary tangles" containing hyperphosphorylated tau protein. AP42 is a peptide containing 42 amino acids, which is physiologically soluble and non-toxic. Aβcan induce neuron apoptosis and loss of synapse. influence the balance of Ca2+, and induce the production of reactive oxygen species (ROS). Aggregation of Aβmonomer into multimeric aggregates has been strongly associated with the neurodegenerative pathology and a cascade of harmful event related to AD. There are soluble monomers, oligomers and insoluble fibrils in AD brains, and soluble Aβoligomers are more toxic in vitro and in vivo than fibrils, and may represent the primary pathological species.Finding molecules to intervene Aβaggregation and toxicity provides an optimum selection for therapeutic target. Recnetly, moderate wine consumption is receiving increasing attention in AD research. Some reports suggest that red wine intake may protect against AD and attenuate AD-type cognitive deterioration and amyloid neuropathology. Red wine contains a broad range of polyphenols that may interact with some peptides and proteins and have wide-ranging properties on preventing and treating various diseases including neuroprotective effects both in vivo and in vitro. Polyphenols have potent anti-amyloidogenic activities. Polyphenols extracted from grape seeds were able to inhibit Aβaggregation, reduced Aβproduction and protected against Aβneurotoxicity in vitro. In present study, the effects of two polyphenols resveratrol and ellagic acid on Aβ42 aggregation and cytotoxicity were elucidated. Moreover, we reported the toxicity and inflammatory response of Aβ1-16.Experimental methods1. Effects of resveratrol on Aβ42 aggregation and cytotoxicity(1) The effects of resveratrol at different concentrations on AP42 aggregation by ThT-induced fluorescence detection(2) The effects of resveratrol on Aβ42 morphologies by TEM observation(3) The effects of resveratrol on AP42 secondary structure by CD measure-ments(4) The effects of resveratrol on AP42 oligomers formation by Elisa and dot-blot detection(5) Disaggregation of Aβ42 fibrils by resveratrol(6) Cytotoxicity of Aβ42 and fAβ42 co-incubated with resveratrol by MTT assay2. Effects of ellagic acid on Aβ42 aggregation and cytotoxicity(1) The effects of ellagic acid on Aβ42 aggregation processes by TEM observation(2) Measurement of AP42 oligomer levels by Elisa and western-blot(3) Conformational changes of Aβ42 by CD measurements(4) Cell viability measurement3. The toxicity and inflammatory responses of Aβ1-16(1) Measurement of Aβ1-16 toxicity by MTT assay (2) Inflammatory factors released by BV-2 glial cells stimulated by Aβ1-16(3) Morris water maze(4) ImmunohistochemistryResults1. Effects of resveratrol on Aβ42 aggregation and cytotoxicity(1) Resveratrol inhibited Aβ42 aggregation as detected by ThT, TEM and CD.(2) Resveratrol did not inhibit Aβ42 oligomer formation. Resveratrol main-tained a higher oligomer level over the test period after a short initial phase while Aβ42 alone showed decreasing signals with incubation proceeding after the oligomerization phase, indicating that some of Aβ42 oligomers in the samples of Aβ42 alone aggregated to fibrils and resveratrol could stabilize the formed Aβ42 oligomers.(3) Resveratrol disaggregated performed AP42 fibrils as shown by ThT, western-blot and TEM.(4) Resveratrol could effectively attenuate the cytotoxicity of Aβ42 towards SH-SY5Y cells.2. Effects of ellagic acid on Aβ42 aggregation and cytotoxicity(1) Ellagic acid accelerated Aβ42 aggregation into fibrils by TEM and western-blot detection.(2) Ellagic acid significantly reduced Aβ42 oligomers levels detected by Elisa.(3)The addition of ellagic acid acceleratedβ-sheet formation as shown by CD results.(4) Ellagic acid reduced Aβ42 cytotoxicity toward SH-SY5Y cells by MTT assay.3. The toxicity and inflammatory responses of Aβ1-16 (1) The morphologies and numbers of SH-SY5Y cells obviously changed after Aβ1-16 (30μM) added for 48 h.(2) The cell viability of SH-SY5Y cells were affected by Aβ1-16 in a dose dependent manner, but not for Aβ1-8.(3) The toxicity of Aβ1-16 decreased after incubation at different temperature (4℃,37℃)for 3dor7d.(4)Aβ1-16 stimulated the secretion of TNF-a, but not IL-1βreleased by BV-2 microglia cells, and 100μM Aβ1-16 significantly increased IL-4 levels.(5) Morris water maze:The latencies and crosses of Aβ1-16 group were no better than the other two groups, which indicated the memory deficiency of Aβ1-16 group.(6) The astrocytes were activated by Aβ1-16 as shown by the results of immunohistochemistry.Conclusions1. Resveratrol inhibited Aβ42 aggregation but did not prevent oligomer formation.2. Resveratrol could disaggregate performed fibrils.3. Resveratrol attenuated Aβ42 cytotoxicity.4. Ellagic acid promoted Aβ42 aggregation with less time of oligomer existence, and inhibited Aβ42 cytotoxicity.5. The above two polyphenols influenced AP42 aggregation in two different pathway, but both alleviated the cytotoxicity. The one reason might attribute to their antioxidant activity, and the other more important one might be the conformation changes of oligomers due to the interaction between the polyphenols and Aβ42.6. Aβ1-16 decreased the viability of SH-SY5Y cells in a dose dependent manner, and stimulated the secretion of TNF-a and IL-4 by BV-2 microglia cells. Injection of Aβ1-16 to hippocampus of C57 mouse led to memory deficiency.更多还原