Aβ-Cu复合物与Aβ纤丝对小胶质细胞激活作用比较研究

目的:比较具有氧化还原活性的过渡金属离子Cu~(2+)(Cu)诱导形成的Aβ聚集物(Aβ-Cu复合物)与Aβ自聚集形成的纤丝(Fibrillar Aβ,f Aβ)对小胶质细胞激活作用的差异。方法:制备Aβ-Cu复合物和fAβ,利用小鼠BV-2小胶质细胞株,分别以不同浓度的Aβ-Cu复合物和fAβ于37℃刺激24 h,检测细胞上清液中的TNF-α(Tumor necrosis factor-α)、NO(Nitric oxide)以及H_2O_2(Hydrogen Peroxide)的含量。分别收集Aβ-Cu复合物和f Aβ作用24 h后的大鼠原代小胶质细胞条件培养液,通过观察该条件培养液对大鼠原代海马神经元细胞活力的影响,评价小胶质细胞介导的间接神经元毒性。结果:(1)在不引起直接神经毒性剂量(2.5μM)下,Aβ-Cu复合物激活小胶质细胞释放TNF-α(P0.01)、NO(P0.05)以及H_2O_2(P0.05)的作用强于f Aβ。(2)在此剂量下,Aβ-Cu复合物通过激活小胶质细胞引起的间接神经元毒性强于fAβ(P0.05)。结论:与fAβ相比,非神经毒性剂量的Aβ-Cu复合物对于小胶质细胞具有更强的激活作用,并由此引发更为明显的神经元毒性。     

PACAP受体激活对抗Aβ25-35致神经元凋亡作用的观察

目的和方法：本研究采用体外原代培养新生大鼠海马神经元的方法探讨了垂体腺苷环化酶激活多肽（PACAP）对抗淀粉样蛋白（Aβ25-35）致凋亡的作用机理。结果：25μmol/L的Aβ蛋白处理神经元5d即有明显的凋亡特征出现。PACAP27（P27）在正常情况下对海马神经元无明显营养作用,但当Aβ导致神经元凋亡时P27有显著的保护作用,可以提高神经元的活性,减少细胞凋亡数目,降低基因组小片段DNA含量。PACAP受体拮抗剂PACAP6－27（P6－27）可以逆转P27的保护作用。结论：研究结果说明PACAP通过受体激活参与对抗Aβ的神经毒性效应。     

β-淀粉样肽诱导小胶质细胞培养上清致海马神经元损伤模型的建立

目的：建立β淀粉样肽（Aβ1-40）诱导激活小胶质细胞的上清致海马神经元损伤的细胞模型,并初步研究神经元损伤的机制。方法：用不同浓度的可溶性Aβ1-40诱导激活小胶质细胞,光镜下观察不同时间点的细胞形态,ELISA检测其分泌的肿瘤坏死因子仪;用激活后的小胶质细胞条件培养基刺激海马神经元,光镜下观察细胞形态,Western blot检测刺激后海马神经元内诱导型一氧化氮合酶（iNOS）和硝基酪氨酸（NT）的表达水平,ELISA检测海马神经元内胱冬蛋白酶-3（caspase-3）活性来评价神经元的损伤程度。结果：终浓度为10μmol／L的Aβ1-40与小胶质细胞孵育24h后,取上清液加到培养的海马神经元,孵育24-72h,海马神经元较对照组形态有明显变化;经Western blot检测,神经元内iNOS、NT表达明显增加;ELISA检测神经元内caspase-3活性明显增高。结论：小胶质细胞被Aβ1-40激活后,其释放物有明显的致神经元损伤效应,表明建立了神经元损伤模型。     

Cu2+-Aβ 复合物与Aβ 单体诱导神经元 H2O2 释放作用的比较研究

目的:比较不同摩尔比Cu~(2+)-Aβ复合物与Aβ单体诱导神经元H_2O_2释放作用的差异。方法:制备不同摩尔比(0.1-5)的Cu~(2+)-Aβ复合物,通过检测硫磺素T(Thioflavin T,Th T)荧光强度考察Cu~(2+)对Aβ纤丝形成的影响。利用原代培养的大鼠海马神经元细胞,分别以不同摩尔比Cu~(2+)-Aβ复合物,不同浓度Cu~(2+)-Aβ复合物(摩尔比为1),以及Aβ单体和Cu~(2+)处理细胞,检测培养上清中的H_2O_2含量;分离线粒体,分别检测不同浓度Cu~(2+)-Aβ复合物(摩尔比为1),以及Aβ单体和Cu~(2+)处理后H_2O_2的释放;观察不同摩尔比Cu~(2+)-Aβ复合物,不同浓度Cu~(2+)-Aβ复合物(摩尔比为1),以及Aβ单体和Cu~(2+)对神经元细胞活力的影响。结果:(1)Th T荧光试验结果表明,Cu~(2+)与Aβ(10μM)摩尔比为1~5范围内可明显抑制Aβ纤丝形成。(2)Cu~(2+)-Aβ复合物(摩尔比为1~5;Aβ浓度为10μM)以及摩尔比为1的Cu~(2+)-Aβ复合物(Aβ浓度分别为5,10μM)可显著诱导神经元释放H_2O_2;另外,摩尔比为1时,Cu~(2+)-Aβ复合物还可诱导神经元线粒体内H_2O_2释放;上述作用均强于Aβ单体或Cu~(2+)。(3)Cu~(2+)-Aβ复合物(摩尔比为1~5)可显著降低神经元细胞活力,该作用强于Aβ单体或Cu~(2+)。结论:与Aβ单体相比,Cu~(2+)-Aβ复合物诱发神经元细胞及其线粒体释放H_2O_2作用更强,并诱发更为明显的神经元毒性。提示Cu~(2+)与Aβ之间的配位结合可能增强其引发活性氧释放以及神经元毒性反应;Cu~(2+)-Aβ复合物引发的活性氧可能主要来自线粒体。     

β淀粉样蛋白1-42对BV-2细胞产生一氧化氮功能的刺激作用

目的应用β淀粉样蛋白1-42(β-Amyloid,Aβ1-42)作用于小胶质细胞(microglia,MG),对MG产生一氧化氮(nitric oxide,NO)的作用进行研究.方法应用高度纯化的BV-2小胶质细胞作为体外小胶质细胞模型,测定加入Aβ1-42后细胞上清NO含量及细胞iNOS酶活力;Western blot法测定Aβ对BV-2细胞iNOS蛋白表达的影响,免疫细胞化学方法对iNOS蛋白的表达情况进行观察.结果 Aβ1-42可以刺激BV-2细胞产生NO、提高细胞iNOS酶活性、增加iNOS蛋白质表达,以上作用均具有时间及浓度依赖性.结论 Aβ1-42在体外可通过提高细胞iNOS酶活性、增加iNOS蛋白质表达而增加NO的分泌,为NO发挥神经元毒性作用创造了条件.     

川芎嗪对Aβ25-35诱导体外大鼠海马神经元细胞凋亡的影响

本文通过Aβ25-35诱导体外原代培养的SD乳大鼠海马神经元,建立Aβ毒性损伤细胞模型,结合AnnexinV-FITC/PI荧光双染法流式细胞术、MTT比色法、实时荧光定量PCR及Western blot方法检测川芎嗪(tetrameth-ylpyrazine,TMP)对原代培养的海马神经元细胞活性、早期凋亡率和Bax、Bcl-2基因表达的影响。结果显示川芎嗪高、中剂量可明显增强细胞活性,增加神经元细胞的存活率(P<0.01),可显著抑制海马神经元细胞早期凋亡(P<0.01),抑制凋亡蛋白Bax的表达(P<0.01),增强抗凋亡蛋白bcl-2的表达(P<0.01)。川芎嗪可通过调节Bax/Bcl-2平衡抵抗Aβ25-35诱导的海马神经元凋亡,降低Aβ的神经元毒性,对海马神经元损伤有明显的保护作用。     

维生素C保护Aβ1-40Cu(Ⅱ)复合物引起的神经元氧化损伤

老年斑中存在大量β 淀粉样蛋白（β-amyloid, Aβ）是老年痴呆症（Alzheimer′s disease, AD）的重要病理特征.大量数据表明,Aβ上具有与过渡态金属离子共价结合的位点,二者能结合成为寡聚复合物. Aβ_1-40Cu（Ⅱ）复合物通过Cu²⁺的还原催化O₂产生H₂O₂但反应机制不清.本文尝试以天然抗氧化剂维生素C（VC）来对抗Aβ_1-40及Aβ_1-40Cu（Ⅱ）复合物产生的H₂O₂对原代培养的神经细胞的毒性.结果表明,VC能够起到显著的保护作用,其有效浓度为1mmol/L.本文用胞外乳酸脱氢酶泄漏量和H₂O₂生成量的数据证实了细胞存活率（MTT实验）的实验结果.这些结果表明,Aβ_1-40Cu（Ⅱ）复合物能够释放更多的H₂O₂,引发细胞膜破裂并最终引起细胞死亡.加入VC后,神经元受到的损伤较轻,提示VC在保护细胞免受氧化损伤方面发挥了重要作用.     

SK3钾通道通过PI3K/Akt-ERK通路介导Cu2+-Aβ复合物所致小胶质细胞激活

摘要 目的：探讨Ca²⁺激活的小电导SK3钾通道在Cu²⁺-Aβ复合物（Cu-Aβ）所致小胶质细胞激活中的作用及下游信号通路。方法：应用Cu-Aβ激活BV2小胶质细胞,采用ELISA和Amplex Red试剂盒检测细胞培养上清中肿瘤坏死因子（TNF-α）和过氧化氢（H₂O₂）的含量,应用qPCR和Western blot检测钾通道mRNA和蛋白水平及相关信号通路蛋白的磷酸化。结果：（1）应用不同离子通道阻断剂以及不同亚型钾通道阻断剂预处理的实验结果表明,SK3通道可能介导了Cu-Aβ所致的小胶质细胞激活。（2）qPCR和Western blot检测结果表明,Cu-Aβ可上调小胶质细胞内SK3 mRNA和蛋白表达。（3）通过转染SK3-siRNA下调小胶质细胞内SK3表达水平,结果表明,下调SK3表达后显著抑制Cu-Aβ所致的小胶质细胞激活。（4）应用特异性信号分子阻断剂预处理的实验结果表明,PI3K/Akt信号和 ERK信号均参与了Cu-Aβ所致的小胶质细胞激活。（5）应用相关信号分子阻断剂预处理的实验结果进一步表明,在介导Cu-Aβ诱发的小胶质细胞激活过程中,SK3通道位于PI3K/Akt-ERK信号通路的上游。结论：SK3通道通过其下游的PI3K/Akt-ERK信号通路介导Cu-Aβ所致的小胶质细胞炎症反应。     

星形胶质细胞在阿尔茨海默病中的功能变化及分子机制

星形胶质细胞在脑内数量最多,分布最广,对神经元有营养支持的作用,并且能够调控神经元的活性。越来越多的证据表明星形胶质细胞激活参与阿尔茨海默病(Alzheimer's disease,AD)的发生和发展。在AD病理情况下,星形胶质细胞在多种因子如β淀粉样蛋白(beta-amyloid,Aβ)和促炎细胞因子的作用下被激活,激活的星形胶质细胞进一步释放一氧化氮(Nitric oxide,NO)和多种炎性因子增强炎症级联反应。功能失常的星形胶质细胞会促进Aβ的产生,减弱对Aβ的摄取和清除,导致Aβ聚集沉积形成老年斑。激活的星形胶质细胞释放的炎症因子还能显著增加神经元内tau蛋白的异常过度磷酸化,产生神经纤维缠结。本文对星形胶质细胞在AD中参与神经变性的功能变化和分子机制进行总结,为星形胶质细胞作为靶点预防及治疗AD提供一定的理论依据。     

小胶质细胞与阿尔茨海默病

国内外对阿尔茨海默病（Alzheimer’s disease,AD）神经元病理和神经胶质细胞病理机制进行了大量探索,小胶质细胞（microglia,MG）是中枢神经系统的免疫细胞,在致炎因素作用下它被激活成反应性MG,反应性MG既具有保护神经元的作用,也能分泌细胞毒因子、补体蛋白而损害神经元。尽管目前AD发病机理还不清楚,但大多数学者认为β淀粉样蛋白（Aβ）沉积激活MG引起的炎症反应是AD的核心病理机制。     

A homozygous female hemophilia A

BACKGROUND:

Hemophilia A (HA), being an X-linked recessive disorder, females are rarely affected, although they can be carriers.

AIMS:

To study the mutation in F8 gene in an extended family with a homozygous female HA.

MATERIALS AND METHODS:

All the seven affected members (six males and one female) were initially screened by Conformation Sensitive Gel Electrophoresis (CSGE) and direct DNA sequencing.

RESULTS:

A homozygous missense mutation c.1315G>A (p.Gly420Ser) was identified in exon 9 of F8 gene in homozygous state in the affected female born of 1° consanguinous marriage and in all the affected male members of the family. Her factor VIII levels was found to be 5.5%, vWF:Ag 120%.

CONCLUSION:

In India, as consanguineous marriages are very common in certain communities (up to 30%), the likelihood of encountering female hemophilia is higher, although this is the first case of HA out of 1600 hemophilia families registered in our Comprehensive Haemophilia Care Center. Genetic diagnosis in such cases is not necessary as all the male children will be affected and daughters obligatory carriers.     

Aminopeptidase A is angiotensinase A

Summary Quantitative histochemical measurements of aminopeptidase A (APA; E.C.3.4.11.7) were done kinetically in the kidney glomeruli of rat and mouse with an instrumental setup consisting of a microdensitometer and a computer-supported morphometric system. The histochemical demonstration of APA was carried out using the simultaneous azo coupling technique (purest-grade Fast Blue B as coupling agent and -l-glutamic acid-4-methoxy-2-naphthylamide as substrate). The methodological studies show that APA activity is calcium-ion-dependent and increases linearly with the thickness of the tissue section (3–12 m) and that the time-course of APA activity as determined by linear regression is linear only for the first 1 to 2 min of the reaction. — Kinetic measurements indicate a 40% decrease in APA activities when -l-glutamic acid-4-methoxy-2-naphthylamide (-l-Glu-MNA) is replaced by -l-aspartic acid-4-methoxy-2-naphthylamide. When -l-Glu-MNA is replaced with l-alanine-4-methoxy-2-naphthylamide, which is a substrate of aminopeptidase M (APM) only very low reaction rates are measurable (about 1.4% of those with -l-Glu-MNA). 100 and 130 mM NaCl in the incubation medium increase APA activities by approximately 16%–17%. — To clarify the functional importance of APA in the kidney, their activities were measured under the influence of angiotensins. The glomerulus was selected as the measuring site, for besides APA it contains no APM or other peptidases that could degrade angiotensins (the glomerular dipeptidyl peptidase IV is not inhibited by angiotensin II). Using the Lineweaver-Burk plot, we determined a K _m of 0.16 mM for the APA in rat glomeruli and 0.14 mM in mouse glomeruli. The V _max in mouse glomeruli is 1.6 times higher than in rat glomeruli. Ang iotensin I, II and III competitively inhibit APA in the rat and mouse glomeruli. — With quantitative histochemical techniques it was possible to show that APA is equivalent to angiotensinase A (splitting off the N-terminal aspartic acid from angiotensin I and II).Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

A glucoside of urospermal A

The aerial parts of Urospermum picroides afforded, in addition to urospermal A a p-hydroxylphenyl acetate of a glucoside of urospermal A.     

Quadruplexes of human telomere DNA analogs designed to contain G:A:G:A,G:G:A:A,and A:A:A:A tetrads

Replacement of two to four guanines by adenines in the human telomere DNA repeat dG₃(TTAG₃)₃ did not hinder the formation of quadruplexes if the substitutions took place in the terminal tetrad bridged by the diagonal loop of the intramolecular antiparallel three‐tetrad scaffold, as proved by CD and PAGE in both Na⁺ and K⁺ solutions. Thermodynamic data showed that, in Na⁺ solution, the dG₃(TTAG₃)₃ quadruplex was destabilized, the least by the two G:A:G:A tetrads, the most by the G:G:A:A tetrad in which the adenosines replaced syn‐guanosines. In physiological K⁺ solution, the highest destabilization was caused by the 4A tetrad. In K⁺, only the unmodified dG₃(TTAG₃)₃ quadruplex rearranged into a K⁺‐dependent quadruplex form, none of the multiple adenine‐modified structures did so. This may imply biological consequences for nonrepaired A‐for‐G mutations. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 880–886, 2010.     

A monoclonal antibody to ochratoxin A

The production of patulin by P. expansum Link ex Gray growing in apple juice was determined over a range of pH. Patulin was found to be produced in large quantities over a narrow range of pH from 3-2 to 3–8. The biomass produced increased with increasing pH. The changes in pH due to growth of the organism were found to be small.