阿尔兹海默病中DNA甲基化对β-淀粉样蛋白的影响

DNA甲基化是最常见也是目前研究得最成熟的表观遗传机制,近年来其在阿尔兹海默病(Alzheimer’s disease,AD)等神经系统退行性疾病中的作用逐渐受到研究者的关注。β-淀粉样蛋白(Beta-amyloid,Aβ)变性聚集与AD的主要病理特征——老年斑(Senile plaques,SP)的形成密切相关。同时,它还能诱导细胞凋亡、激发炎症级联反应、产生氧化应激、导致线粒体功能障碍等,从而加剧AD的病理过程。研究表明,DNA甲基化在Aβ生成、清除及毒性相关基因的调控中发挥着重要作用。由于DNA甲基化的可逆性,深入探究DNA甲基化在AD发生、发展中的作用,可能为AD的治疗带来曙光。文章综述了AD中DNA甲基化对Aβ的影响,进一步阐释了AD发病的表观遗传学机制,为AD的表观遗传学治疗提供了理论基础。     

脑源性神经营养因子拮抗β淀粉样蛋白所致大鼠在体海马长时程增强的伤害

目的:探讨脑源性神经营养因子(BDNF)对β淀粉样蛋白(Aβ)致大鼠突触功能障碍的保护作用。方法:36只健康雄性SD大鼠随机分为对照、Aβ25-35、BDNF、不同剂量BDNF(0.02μg,0.1μg,0.5μg)+Aβ25-35等六组(n=6)。实验采用电生理学手段,利用自制的海马给药装置和刺激/记录绑定电极引导和记录大鼠在体海马CA1区场兴奋性突触后电位(fEPSPs)和高频刺激(HFS)诱导的长时程增强(LTP)。结果:①海马CA1区注射Aβ25-35(2 nmol)不影响基础性fEPSPs,但能显著抑制LTP的诱导与维持,HFS后fEPSPs平均幅度较对照组明显降低(P<0.01);②海马CA1区注射BDNF(0.1μg)不影响基础性fEPSPs,也不影响LTP的诱导与维持,HFS后fEPSPs平均幅度与对照组相比没有明显差异(P>0.05);③与单独给予Aβ25-35相比,不同浓度的BDNF(0.1μg,0.5μg)与Aβ25-35合用组在HFS后0 min、30 min和60 min时的fEPSPs平均幅度均明显增加(P<0.01),并具有一定的剂量依赖性,表明BDNF预处理可有效拮抗Aβ25-35引起的LTP抑制。结论:脑内注射BDNF能够预防和拮抗由Aβ25-35引起的海马LTP损伤,提示BDNF水平的上调有助于维持正常的突触可塑性并可能改善AD患者的学习记忆功能。     

β-淀粉样蛋白对BV2小胶质细胞促炎作用的研究

目的探讨β-淀粉样蛋白(β-amyloid,Aβ)促进BV2小胶质细胞产生炎性因子IL-1β和TNFα的作用机制。方法体外培养BV2小胶质细胞,应用Aβ1-42作用于BV2小胶质细胞,或用吡咯烷二硫代氨基甲酸盐(PDTC)预孵育再给予Aβ1-42刺激,实时荧光定量反转录聚合酶链反应法(RT–PCR)检测IL-1β和TNFαmRNA表达;免疫印迹法(Western blot)检测胞核中NF-κB p65及其抑制蛋白胞浆中IkBα的表达。结果 Aβ1-42作用于BV2小胶质细胞后,Westernblot显示胞浆内IkBα表达下降,胞核内NF-κB p65表达明显增加,RT-PCR测定IL-1β和TNFαmRNA的表达增加;给予NF-κB信号通路特异阻断剂PDTC后,胞浆IkBα的下降和胞核内NF-κB p65的增加均被抑制,同时IL-1β和TNFαmRNA的表达亦受到抑制,PDTC的抑制效果呈剂量依赖性。结论 Aβ可通过激活小胶质细胞NF-κB信号通路促进IL-1β和TNFα的表达。     

田蓟苷对脑小血管病大鼠模型认知功能受损和神经细胞凋亡的影响

摘要 目的：探讨田蓟苷对脑小血管病（cerebral small vessel disease,CSVD）大鼠模型认知功能受损和神经细胞凋亡的影响及机制。方法：将SD大鼠分为Sham组、CSVD组、低剂量田蓟苷组（L-Til组,5 mg/kg/d）、中剂量田蓟苷组（M-Til组,10 mg/kg/d）和高剂量田蓟苷组（H-Til组,20 mg/kg/d）。通过同种系微栓子体外注入法建立CSVD大鼠模型,各组大鼠均治疗4周。治疗结束后对各组大鼠进行Morris水迷宫测试,分离海马组织并进行HE染色、TUNEL染色和尼氏染色。通过免疫组化染色或Western blot检测大鼠海马组织中Bax、Bcl2、cleaved caspase-3、VEGF和细胞核NF-κB p65的表达。使用相应试剂盒检测血清炎症指标（TNF-α和IL-1β）和氧化应激指标（SOD和MDA）水平。结果：与CSVD组比较,L-Til组、M-Til组和H-Til组的逃避潜伏期均缩短,而穿越平台次数均增加（P<0.05）。与CSVD组比较,L-Til组、M-Til组和H-Til组大鼠海马组织TUNEL阳性率降低,而尼氏体光密度增加（P<0.05）;Bax和cleaved caspase-3的表达水平降低,Bcl2水平升高（P<0.05）。与CSVD组比较,L-Til组、M-Til组和H-Til组大鼠血清中TNF-α、IL-1β和MDA水平降低,SOD升高（P<0.05）。与CSVD组比较,L-Til组、M-Til组和H-Til组大鼠海马组织中细胞核NF-κB p65蛋白表达水平降低,细胞质VEGF蛋白表达水平升高（P<0.05）。结论：本研究证明田蓟苷可减轻CSVD大鼠的认知功能障碍并抑制神经细胞凋亡,田蓟苷对CSVD的治疗机制部分通过减少炎症和氧化应激、促进VEGF的表达和抑制NF-κB信号通路来实现。     

淀粉样蛋白A、D-二聚体、肌酸激酶同工酶联合检测对川崎病患儿冠状动脉损伤的诊断价值分析

摘要 目的：探讨血清淀粉样蛋白A(SAA)、D-二聚体(D-D)、肌酸激酶同工酶(CK-MB)联合检测对川崎病患儿冠状动脉损伤(CAL)的诊断价值。方法：选取2018年9月～2021年5月我院收治的80例川崎病患儿,根据是否合并CAL分为CAL组(n=34)和NCAL组(n=46)。收集患儿基础资料,并检测SAA、D-D、CK-MB水平。多因素Logistic回归分析川崎病患儿CAL影响因素,受试者工作特征（ROC）曲线分析血清SAA、D-D、CK-MB水平对川崎病患儿CAL的诊断价值。结果：与NCAL组比较,CAL组C反应蛋白(CRP)、红细胞沉降率(ESR)、SAA、D-D、CK-MB水平升高(P＜0.05)。多因素Logistic回归分析显示,CRP、ESR、SAA、D-D、CK-MB为川崎病患儿CAL独立影响因素(P＜0.05)。SAA、D-D、CK-MB、三项联合诊断川崎病患儿CAL的曲线下面积（AUC）分别为0.661、0.687、0.746、0.799,联合应用的诊断效能最高。结论：血清SAA、D-D、CK-MB是川崎病患儿CAL独立影响因素,且联合检测以上指标可辅助诊断川崎病患儿CAL。     

血清BNP、h-FABP对川崎病患儿的诊断价值分析

目的:探讨与分析血清脑钠肽(Brain natriuretic peptide,BNP)、心肌型脂肪酸结合蛋白(heart type fatty acid binding protein,H-FABP)对川崎病(Kawasaki disease,KD)患儿的诊断价值。方法:采用病例对照的方法,选择2018年1月至2019年7月在本院诊治住院的川崎病患儿78例作为川崎病组,选取同期体检的健康儿童78例作为对照组。调查两组的临床资料,检测血清BNP、h-FABP水平并进行诊断价值判断。结果:两组的一般资料无统计学意义(P0.05)。川崎病组的白细胞计数、血小板计数、淋巴细胞计数显著高于对照组,红细胞比容与血红蛋白值显著低于对照组(P0.05)。川崎病组患儿的血清BNP、h-FABP含量都显著高于对照组(P0.05)。在川崎病组中,Spearman等级相关性分析显示BNP与h-FABP呈显著正相关性(r=0.782,P=0.000)。受试者工作特征曲线(receiver operating characteristic curve,ROC)显示BNP与h-FABP诊断川崎病的曲线下面积分别为0.822、0.845,诊断灵敏性、特异性与准确性都在70.0%以上。结论:血清BNP、h-FABP在川崎病患儿中呈现高表达状况,对川崎病也有很好的诊断价值,可为临床诊治起到一定的指导作用。     

功能性核磁共振成像技术对原发性三叉神经痛患者脑功能的评估价值及血清IL-1β、TNF-α检测的临床意义

目的:研究功能性核磁共振成像(fMRI)技术对原发性三叉神经痛(ITN)患者静息状态下脑功能的评估价值及血清白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)检测的临床意义。方法:选取2017年2月～2019年2月安徽医科大学附属口腔医院接受诊治的ITN患者共50例进行研究,记为ITN组。另选取同期接受体检的健康人员50例记作健康对照组。两组均于静息状态下行fMRI扫描,比较两组脑区的平均低频振幅率(mfALFF)以及血清IL-1β、TNF-α水平。结果:ITN组左侧的枕中回、枕下回、梭状回、距状裂周围皮层、中央旁回、三角部额下回、右侧背外侧额上回以及左小脑脚1区的mfALFF值高于健康对照组(均P0.05)。ITN组右颞上回、右颞中回、右颞下回、右中央沟盖、右缘上回、右岛盖部颞下回、左前扣带回、扣带旁回、左颞极、颞上回脑区的mfALFF值显著低于健康对照组(均P0.05)。ITN组患者的血清IL-1β、TNF-α水平高于健康对照组(均P0.05)。结论:fMRI对ITN患者静息状态下脑功能的评估价值较高,血清IL-1β、TNF-α水平升高可能与ITN的发病密切相关,可发挥促进疾病发生、发展的作用。     

血清脑钠肽、超敏C反应蛋白、可溶性ST2对阵发性心房颤动患者射频消融术后复发的预测价值研究

摘要 目的：探讨血清脑钠肽（BNP）、超敏C反应蛋白（hs-CRP）、可溶性致瘤抑制素2（sST2）对阵发性心房颤动（AF）患者射频消融（RFA）术后复发的预测价值。方法：选择2016年1月至2020年12月我院收治的接受RFA术治疗的82例阵发性AF患者,术后随访12个月,根据术后是否复发分为复发组（25例）和未复发组（57例）。检测患者血清BNP、hs-CRP、sST2水平,收集临床相关资料,采用多因素Logistic回归模型分析影响阵发性AF患者RFA术后复发的因素,采用受试者工作特征（ROC）曲线分析血清BNP、hs-CRP、sST2预测阵发性AF患者RFA术后复发的价值。结果：复发组血清BNP、hs-CRP、sST2水平高于未复发组（P＜0.05）。血清BNP、hs-CRP、sST2水平升高、AF病程增长是影响阵发性AF患者RFA术后复发的危险因素（P＜0.05）。血清BNP、hs-CRP、sST2预测阵发性AF患者消融术后复发的曲线下面积分别为0.720、0.694、0.718,联合三者预测阵发性AF患者RFA术后复发的曲线下面积为0.866,高于BNP、hs-CRP、sST2单独预测。结论：阵发性AF患者血清BNP、hs-CRP、sST2水平升高是RFA术后复发的危险因素,联合检测血清BNP、hs-CRP、sST2水平有助于预测阵发性AF患者RFA术后复发。     

Endothelial expression of human amyloid precursor protein leads to amyloid β in the blood and induces cerebral amyloid angiopathy in knock-in mice

The deposition of amyloid β (Aβ) in blood vessels of the brain, known as cerebral amyloid angiopathy (CAA), is observed in most patients with Alzheimer’s disease (AD). Compared with the pathology of CAA in humans, the pathology in most mouse models of AD is not as evident, making it difficult to examine the contribution of CAA to the pathogenesis of AD. On the basis of biochemical analyses that showed blood levels of soluble amyloid precursor protein (APP) in rats and mice were markedly lower than those measured in human samples, we hypothesized that endothelial APP expression would be markedly lower in rodents and subsequently generated mice that specifically express human WT APP (APP770) in endothelial cells (ECs). The resulting EC-APP770⁺ mice exhibited increased levels of serum Aβ and soluble APP, indicating that endothelial APP makes a critical contribution to blood Aβ levels. Even though aged EC-APP770⁺ mice did not exhibit Aβ deposition in the cortical blood vessels, crossing these animals with APP knock-in mice (App^NL-F/NL-F) led to an expanded CAA pathology, as evidenced by increased amounts of amyloid accumulated in the cortical blood vessels. These results highlight an overlooked interplay between neuronal and endothelial APP in brain vascular Aβ deposition. We propose that these EC-APP770⁺:App^NL-F/NL-F mice may be useful to study the basic molecular mechanisms behind the possible breakdown of the blood–brain barrier upon administration of anti-Aβ antibodies.     

Down syndrome,Alzheimer disease,and cerebral amyloid angiopathy: The complex triangle of brain amyloidosis

Down syndrome (DS) is the main genetic cause of intellectual disability worldwide. The overexpression of the Amyloid Precursor Protein, present in chromosome 21, leads to β‐amyloid deposition that results in Alzheimer disease (AD) and, in most cases, also to cerebral amyloid angiopathy (CAA) neuropathology. People with DS invariably develop the neuropathological hallmarks of AD at the age of 40, and they are at an ultra high risk for suffering AD‐related cognitive impairment thereafter. In the general population, cerebrovascular disease is a significant contributor to AD‐related cognitive impairment, while in DS remains understudied. This review describes the current knowledge on cerebrovascular disease in DS and reviews the potential biomarkers that could be useful in the future studies, focusing on CAA. We also discuss available evidence on sporadic AD or other genetically determined forms of AD. We highlight the urgent need of large biomarker‐characterized cohorts, including neuropathological correlations, to study the exact contribution of CAA and related vascular factors that play a role in cognition and occur with aging, their characterization and interrelationships. DS represents a unique context in which to perform these studies as this population is relatively protected from some conventional vascular risk factors and they develop significant CAA, DS represents a particular atheroma‐free model to study AD‐related vascular pathologies. Only deepening on these underlying mechanisms, new preventive and therapeutic strategies could be designed to improve the quality of life of this population and their caregivers and lead to new avenues of treatment also in the general AD population.     

Immunohistochemical characteristics of the constituents of senile plaques and amyloid angiopathy in aged cynomolgus monkeys

Abstract: In this study, we immunohistochemically examined the several constituents of senile plaques (SPs) and cerebral amyloid angiopathy (CAA) in aged cynomolgus monkeys. Apolipoprotein E (apoE) deposited in all mature plaques and CAA, and in half of the diffuse plaques. Alpha-1-antichymotripsin (αACT) deposited in half of the mature plaques and in one third of the CAA. Amyloid precursor protein (APP), ubiquitin (Ub), and microtubule-associated protein-2 (MAP-2) accumulated in the swollen neurites of mature plaques. Glial fibrillary acidic protein (GFAP) was detected in the astrocytes and their processes surrounding the mature plaques. Tau was detected in neither the SPs nor CAA. Therefore, mature plaques involved extracellular Aβ, apoE, and αACT, and also astrocytes and swollen neurites. However, diffuse plaques involved only extracellular Aβ and apoE. Since these features, except for tau, were consistent with those in humans, this animal model will be useful for studying the pathogenesis of cerebral amyloid deposition.     

SAA和CRP联合检测在小儿感染性疾病鉴别诊断中的应用价值

目的探讨血清淀粉样蛋白A(SAA)和C-反应蛋白(CRP)联合检测在小儿感染性疾病鉴别诊断中的临床价值。方法选取2012年1月至2013年12月间住院患儿166例,分为细菌感染组72例,病毒感染组94例,采用免疫比浊法检测急性期和恢复期水平,并与30例健康儿童进行比较分析。结果细菌感染组SAA和CRP浓度显著高于病毒感染组和对照组,病毒感染组SAA显著高于对照组(P0.05)。细菌感染组中重症组SAA和CRP浓度显著高于轻症组,轻症组SAA和CRP浓度显著高于对照组(P0.05)。SAA和CRP单独检测阳性率较低,两者联合检测能提高检出率。结论 SAA和CRP联合检测在小儿感染性疾病鉴别诊断、疗效动态观察中有重要应用价值。     

Cross-seeding of wild-type and hereditary variant-type amyloid beta-proteins in the presence of gangliosides

We investigated the molecular mechanism underlying the ganglioside-induced initiation of the assembly of wild and hereditary variant-type amyloid beta-proteins, including Arctic-, Dutch-, and Flemish-type amyloid beta-proteins. We monitored the assembly of amyloid beta-protein by thioflavin-T assay, western blotting and electron microscopy. We also examined how externally added amyloid beta-protein assembles in a cell culture. The assembly of wild-, Arctic-, Dutch-, and Flemish-type amyloid beta-proteins were accelerated in the presence of GM1, GM1, GM3 and GD3 gangliosides. Notably, all of these amyloid beta-proteins accelerated the assembly of different type of amyloid beta-protein, following prior binding to a specific ganglioside. A specific-ganglioside-bound form of variant-type amyloid beta-protein was recognized by the antibody (4396C) specific to the GM1-ganglioside-induced altered conformation of wild-type amyloid beta-protein. Moreover, the assembly of these amyloid beta-proteins in the presence of a specific ganglioside was markedly suppressed by coincubation with 4396C. This study suggests that cross-seeding can occur between wild and hereditary variant-type amyloid beta-proteins despite differences in their amino acid sequences.     

Mixtures of wild-type and a pathogenic (E22G) form of Abeta40 in vitro accumulate protofibrils,including amyloid pores

Although APP mutations associated with inherited forms of Alzheimer's disease (AD) are relatively rare, detailed studies of these mutations may prove critical for gaining important insights into the mechanism(s) and etiology of AD. Here, we present a detailed biophysical characterization of the structural properties of protofibrils formed by the Arctic variant (E22G) of amyloid-beta protein (Abeta40(ARC)) as well as the effect of Abeta40(WT) on the distribution of the protofibrillar species formed by Abeta40(ARC) by characterizing biologically relevant mixtures of both proteins that may mimic the situation in the heterozygous patients. These studies revealed that the Arctic mutation accelerates both Abeta oligomerization and fibrillogenesis in vitro. In addition, Abeta40(ARC) was observed to affect both the morphology and the size distribution of Abeta protofibrils. Electron microscopy examination of the protofibrils formed by Abeta40(ARC) revealed several morphologies, including: (1) relatively compact spherical particles roughly 4-5 nm in diameter; (2) annular pore-like protofibrils; (3) large spherical particles 18-25 nm in diameter; and (4) short filaments with chain-like morphology. Conversion of Abeta40(ARC) protofibrils to fibrils occurred more rapidly than protofibrils formed in mixed solutions of Abeta40(WT)/Abeta40(ARC), suggesting that co-incubation of Abeta40(ARC) with Abeta40(WT) leads to kinetic stabilization of Abeta40(ARC) protofibrils. An increase in the ratio of Abeta(WT)/Abeta(MUT(Arctic)), therefore, may result in the accumulation of potential neurotoxic protofibrils and acceleration of disease progression in familial Alzheimer's disease mutation carriers.     

Generation of amyloid beta protein from a presenilin-1 and betaAPP complex

Presenilin-1 (PS1) is a causative gene in early onset familial Alzheimer's disease (FAD). FAD-linked mutant PS1s significantly increased Abeta40 and Abeta42(43) levels (P < 0.001) and decreased the production of an 11.4 kD (beta-stub) and an 8.7 kD (alpha-stub) carboxyl-terminal fragment of amyloid beta precursor protein (betaAPP-CTFs) (P < 0.01). In the 2% CHAPS extracted lysates, the complex containing the amino-terminal fragment of PS1 (PS1-NTF), the carboxyl-terminal fragments of PS1 (PS1-CTF), and betaAPP-CTFs was identified. Incubation of this isolated complex at pH 6.4 showed the direct generation of Abeta40 and gamma-stub from this complex. This reaction was inhibited by a gamma-secretase inhibitor. The degrading rate of a co-precipitated beta-stub was facilitated under the presence of FAD-linked mutant PS1s. This findings suggest that the direct generation of Abeta from the complex may play an important role in the pathogenesis of Alzheimer's disease.     

Ultrastructure and pathology of prion protein amyloid accumulation and cellular damage in extraneural tissues of scrapie-infected transgenic mice expressing anchorless prion protein

In most human and animal prion diseases the abnormal disease-associated prion protein (PrPSc) is deposited as non-amyloid aggregates in CNS, spleen and lymphoid organs. In contrast, in humans and transgenic mice with PrP mutations which cause expression of PrP lacking a glycosylphosphatidylinositol (GPI)-anchor, most PrPSc is in the amyloid form. In transgenic mice expressing only anchorless PrP (tg anchorless), PrPSc is deposited not only in CNS and lymphoid tissues, but also in extraneural tissues including heart, brown fat, white fat, and colon. In the present paper, we report ultrastructural studies of amyloid PrPSc deposition in extraneural tissues of scrapie-infected tg anchorless mice. Amyloid PrPSc fibrils identified by immunogold-labeling were visible at high magnification in interstitial regions and around blood vessels of heart, brown fat, white fat, colon, and lymphoid tissues. PrPSc amyloid was located on and outside the plasma membranes of adipocytes in brown fat and cardiomyocytes, and appeared to invaginate and disrupt the plasma membranes of these cell types, suggesting cellular damage. In contrast, no cellular damage was apparent near PrPSc associated with macrophages in lymphoid tissues and colon, with enteric neuronal ganglion cells in colon or with adipocytes in white fat. PrPSc localized in macrophage phagolysosomes lacked discernable fibrils and might be undergoing degradation. Furthermore, in contrast to wild-type mice expressing GPI-anchored PrP, in lymphoid tissues of tg anchorless mice, PrPSc was not associated with follicular dendritic cells (FDC), and FDC did not display typical prion-associated pathogenic changes.