老年小鼠海马CA1区轴棘突触微细结构的定量分析

目的观察老年小鼠海马CA1区轴棘突触相关结构的改变,为衰老引起的学习记忆减退提供神经解剖学依据。方法利用Golgi染色、超薄连续切片及NIH图像分析系统测量海马CA1区锥体细胞树突棘的密度、树突棘头及突触后致密斑的大小。结果海马CA1第2、3级顶树突的树突棘密度在3月龄组与22月龄组分别为(1.056±0.049)/μm和(0.868±0.038)/μm;穿孔型突触的比率3月龄与22月龄组分别为12.7%和19%。结论老年小鼠海马CA1区锥体细胞树突棘的减少、穿孔型突触比率的增加可能是衰老引起学习记忆减退的形态学基础。     

APP/PS1小鼠海马结构星形胶质细胞胶质纤维酸性蛋白表达及亚甲蓝对其改变的影响

目的 观察APP/PS1转基因小鼠胶质纤维酸性蛋白(GFAP)在海马结构3个亚区星形胶质细胞内的表达变化,同时对比观察亚甲蓝对其表达改变的影响.方法 20只3月龄APP/PS1小鼠,随机分2组,每组10只,对照组:自由饮水;治疗组:根据小鼠饮水量将亚甲蓝加入日常饮水中(25 mg·kg-1 ·d-1)连用4个月至7个月龄.结果 免疫组织化学结果表明治疗组APP/PS1转基因小鼠海马结构3个亚区星形胶质细胞GFAP的表达与对照组相比下调(P＜0.05).Western印迹检测结果显示,治疗组小鼠海马结构3个亚区星形胶质细胞GFAP表达明显弱于对照组(P＜0.05).结论 亚甲蓝可以下调APP/PS1转基因小鼠海马结构3个亚区星形胶质细胞GFAP的表达.     

糖尿病小鼠学习记忆能力减退与海马锥体细胞树突棘变化的相关性

目的探讨代谢异常导致的海马区树突棘变化与认知功能障碍的关系。方法通过水迷宫实验检测动物学习记忆变化,通过Gol-gi-Cox染色结合体视学半定量技术分析海马CA1区锥体细胞树突棘密度变化。结果 2型糖尿病(T2DM)小鼠在一个月出现胰岛素抵抗,并伴随持续的高血糖及高胆固醇血症。2个月出现学习记忆能力下降(P<0.05),随时间延长而加重,4个月后海马CA1区锥体细胞树突棘密度显著下降(P<0.01)。结论研究结果表明T2DM持续高血糖,高血脂可能直接导致海马区神经细胞树突棘丢失,突触可塑性下降,从而出现认知功能障碍。     

姜黄素对APP/PS1双转基因小鼠脑葡萄糖转运子表达的影响

目的采用Morris水迷宫检测小鼠空间学习记忆和记忆保持能力,评价姜黄素对APP/PS1双转基因小鼠认知功能的影响,并观察姜黄素对APP/PS1双转基因小鼠以及海马葡萄糖转运子1(GLUT1)和GLUT3表达的影响,从脑能量代谢的角度探讨姜黄素神经保护作用的机制。方法将3月龄APP/PS1双转基因小鼠随机分为模型(APP/PS1+VEH)组,罗格列酮(APP/PS1+RSG)组,姜黄素大(APP/PS1+curcumin-H)、中(APP/PS1+curcumin-M)、小剂量(APP/PS1+curcumin-L)组。灌胃3个月后,应用免疫组织化学和Western blot方法进行检测。结果 Morris水迷宫撤台实验中,与正常对照(NC)组比较,APP/PS1+VEH组穿越平台次数减少,且目标象限停留时间缩短(P0.01);与APP/PS1+VEH组相比,APP/PS1+curcumin-M组穿越平台次数增加(P0.01)。免疫组织化学染色,与APP/PS1+VEH组相比,APP/PS1+curcumin-H、APP/PS1+curcumin-M组海马CA1区GLUT1阳性细胞增加(P0.05);APP/PS1+curcumin-M组GLUT3阳性细胞计数明显增加(P0.01),APP/PS1+curcumin-H组GLUT3阳性细胞增加(P0.05)。Western blot结果与免疫组织化学结果基本一致。结论姜黄素可改善APP/PS1双转基因小鼠的空间学习记忆和记忆保持能力,影响脑葡萄糖代谢有关的GLUT1和GLUT3蛋白的表达而发挥神经保护作用。     

游泳运动对β淀粉样前体蛋白/早老素1小鼠海马紧密连接蛋白表达和学习记忆的影响

目的 探讨游泳运动对β淀粉样前体蛋白(APP)/早老素1(PS1)双转基因小鼠闭锁小带蛋白1(ZO-1)、闭合蛋白(occludin)表达和学习记忆的影响。方法 将20只APP/PS1小鼠随机分为模型组和游泳组每组10只,对照组选取同窝阴性小鼠10只。游泳组采用游泳运动干预,用新物体识别实验、免疫蛋白印迹法以及免疫荧光检测小鼠学习记忆能力、海马紧密连接蛋白ZO-1、occludin表达和海马CA1区神经元表达情况;硫黄素S染色法检测海马β淀粉样蛋白(Aβ)斑块沉积情况。结果 与对照组比较,模型组辨别指数(RI)明显降低(P<0.01);与模型组比较,游泳组RI明显升高[(63.63±13.35)%vs(50.38±9.83)%,P<0.01]。与对照组比较,模型组ZO-1、occludin明显降低(P<0.05,P<0.01);与模型组比较,游泳组ZO-1、occludin明显增加(0.81±0.02 vs 0.38±0.08,1.07±0.03 vs 0.68±0.12,P<0.05)。与对照组比较,模型组神经元阳性表达明显减少(P<0.01);与...     

NAPP/PS1双转基因小鼠行为学表现及远志皂苷对其调控作用

目的观察APP/PS1双转基因小鼠记忆功能障碍的行为学表现和远志皂苷对其学习记忆能力的保护作用。方法将3月龄APP/PS1双转基因小鼠随机分为模型组、盐酸多奈哌齐组、TEN低、中、高剂量组(18.5、37、74 mg·kg-1·d-1),选同月龄C57BL/6小鼠为对照组。采用Morris水迷宫法和新物体识别试验检测小鼠的学习记忆能力,采用实时定量PCR及Western印迹方法检测海马神经元Bcl-2、Bax和Caspase-3表达水平。结果与模型组相比,TEN各剂量组小鼠学习记忆能力明显改善(P<0.05);与模型组相比,TEN各剂量组Bax和Caspase-3表达水平明显下降(P<0.05),而Bcl-2表达水平显著升高(P<0.05)。结论 TEN可改善小鼠的学习记忆功能,其途径可能是通过上调Bcl-2表达,下调Bax表达,调节Bcl-2/Bax的平衡比值,降低Caspase-3表达来实现。     

姜黄素对APP／PS1双转基因小鼠脑葡萄糖转运子表达的影响

目的采用Morris水迷宫检测小鼠空间学习记忆和记忆保持能力,评价姜黄素对APP／PS1双转基因小鼠认知功能的影响,并观察姜黄素对APP／PS1双转基因小鼠以及海马葡萄糖转运子1（GLUTl）和GLUT3表达的影响,从脑能量代谢的角度探讨姜黄素神经保护作用的机制。方法将3月龄APP／PS1双转基因小鼠随机分为模型（APP／PS1＋VEH）组,罗格列酮（APP／PS1＋RSG）组,姜黄素大（APP／PS1＋curcumin-H）、中（APP／PS1＋curcumin-M）、小剂量（APP／PS1＋curcumin-L）组。灌胃3个月后,应用免疫组织化学和Western blot方法进行检测。结果Morris水迷宫撤台实验中,与正常对照（Nc）组比较,APP／PS1＋VEH组穿越平台次数减少,且目标象限停留时间缩短（P〈0．01）;与APP／PS1＋VEH组相比,APP／PS1＋curcuminM组穿越平台次数增加（P〈0．01）。免疫组织化学染色,与APP／PS1＋VEH组相比,APP／PS1＋curcumin-H、APP／PS1＋curcumin-M组海马CA1区GLUT1阳性细胞增加（P〈0．05）;APP／PS1＋curcumin-M组GLUT3阳性细胞计数明显增加（P〈0．01）,APP／PS1＋curcumin-H组GLUT3阳性细胞增加（P〈0．05）。Westernblot结果与免疫组织化学结果基本一致。结论姜黄素可改善APP／PS1双转基因小鼠的空间学习记忆和记忆保持能力,影响脑葡萄糖代谢有关的GLUT1和GLUT3蛋白的表达而发挥神经保护作用。     

唑尼沙胺对PS1/APP小鼠学习记忆功能和前额叶皮层中BDNF和TrkB的影响

目的探讨唑尼沙胺对PS1/APP小鼠学习记忆功能及前额叶皮层脑源性神经营养因子(BDNF)和酪氨酸激酶受体(TrkB)影响。方法将21只小鼠分为三组,其中14只PS1/APP小鼠随机平均分为PS1/APP生理盐水(PS1/APP-veh)组和PS1/APP唑尼沙胺(PS1/APP-ZNS)组,7只野生C57/BL6J小鼠作为野生生理盐水(WT-veh)组;加药4 w后,对各组小鼠进行Morris水迷宫测试,检测学习记忆功能的变化,利用免疫组化、Western印迹检测前额叶皮层中BDNF和TrkB的表达情况。结果在Morris水迷宫测试中,在唑尼沙胺干预下,PS1/APP小鼠学习记忆功能得到改善;在免疫组化实验和Western印迹实验中,与WT-veh组相比,PS1/APP-veh组BDNF和TrkB表达减少(P<0.05),与PS1/APP-veh组相比,PS1/APP-ZNS组BDNF和TrkB的表达增加(P<0.05)。结论唑尼沙胺可以通过促进前额叶皮层内BNDF和TrkB的表达,改善PS1/APP小鼠中的学习和记忆功能。     

JWH-015预防给药对APP/PS1转基因小鼠学习记忆能力、神经源性炎症和神经可塑性的影响

目的探讨大麻素2型受体(CB2R)激动剂JWH-015预防给药对阿尔茨海默病(AD)模型小鼠空间认知功能及对海马脑区神经源性炎症和神经可塑性的影响。方法 20只6月龄健康APP/PS1转基因AD模型小鼠和20只同窝野生型(WT)小鼠随机分为溶剂对照组(WT+VEH)、AD模型组(APP/PS1+VEH)、JWH-015处理组(APP/PS1+JWH-015)和JWH-015单药组(WT+JWH-015),各组小鼠分别腹腔注射8 w JWH-015 0.5 mg·kg~(-1)·d~(-1)或等量溶剂。通过Morris水迷宫实验检测小鼠空间学习记忆能力;通过刚果红染色检测脑内β淀粉样蛋白(Aβ)斑块的沉积数量;采用免疫荧光组织化学方法观察海马脑区星形胶质细胞标志物胶质纤维酸性蛋白(GFAP)和小胶质细胞标志物离子型钙结合受体分子(Iba)1的免疫荧光变强度及神经源性炎症变化;通过高尔基染色观察海马齿状回(DG)颗粒细胞树突棘突密度检测神经可塑性变化。结果与溶剂对照组相比,AD模型组小鼠Morris水迷宫第5天潜伏期显著延长,第6天穿台次数显著减少(P0.05);同时,海马脑区内出现刚果红染色呈阳性的斑块,胶质细胞标志物GFAP和Iba1免疫荧光强度显著增加,DG区颗粒细胞树突棘突密度显著降低(P0.05)。与AD模型组相比,JWH-015处理组小鼠第5天潜伏期显著降低(P0.05),而第6天穿台次数差异无统计学意义(P0.05);同时,海马脑区Iba1免疫荧光强度显著下降(P0.05),但海马脑区内Aβ斑块沉积数量、GFAP免疫荧光强度及DG区颗粒细胞棘突密度差异无统计学意义(P0.05)。结论 AD模型小鼠空间学习记忆能力损伤可能与海马脑区内Aβ斑块沉积导致的慢性神经源性炎症加重及神经可塑性下降有关;CB2R激动剂预防给药明显改善AD模型小鼠脑内小胶质细胞介导的炎症反应,但对脑内Aβ斑块沉积数量和神经可塑性的下降无影响。     

电针对拟老年性痴呆大鼠学习记忆功能和海马CA1区突触可塑性的影响

目的 探讨电针对拟老年性痴呆(AD)大鼠学习记忆功能和海马CA1区突触可塑性的影响.方法 采用腹腔注射D-半乳糖、东莨菪碱,胃饲三氯化铝(AlCl3)制备多因素损伤拟AD动物模型,在造模同时给予电针干预,应用跳台实验检测大鼠学习和记忆能力,HE染色观察海马CA1区形态学变化,免疫组化测定海马区CA1区突触素表达水平,透射电镜测定突触面数密度、面积密度和体积密度变化.结果 电针能明显改善拟AD大鼠学习记忆能力,增加海马CA1区神经元数目,上调突触素表达.结论 ①多因素损伤能成功制备拟AD大鼠动物模型.②电针促进海马CA1区重建而提高突触素表达可能是改善拟AD鼠的学习记忆功能的机制之一.     

1-Naphthyl isocyanate and 1-naphthylamine as metabolites of 1-naphthylisothiocyanate

Abstract: The importance of the bioactivation of 1-naphthylisothiocyanate was studied. Forty minutes after 1-naphthylisothiocyanate administration to rats, bile was collected over a 2.5-h period; the liver was then excised and homogenized. 1-naphthylisothiocyanate and its metabolites in bile and liver of rats were identified and quantified using coupled gas chromatography-mass spectrometry. Three main compounds were found in all 1-naphthylisothiocyanate-treated animals. They were identified as 1-naphthyl isocyanate, 1-naphthylamine and the parent compound, 1-naphthylisothiocyanate. When rats were given cycloheximide, which attenuates 1-naphthylisothiocyanate toxicity, 30 min before 1-naphthylisothiocyanate (300 mg/kg), 1-naphthyl isocyanate concentration was significantly lower than in rats receiving only 1-naphthylisothiocyanate. The appearance of 1-naphthylamine was also inhibited by cycloheximide, although not to the same extent as 1-naphthyl isocyanate. On the other hand, phenobarbital, which potentiates 1-naphthylisothiocyanate hepatotoxicity, enhanced 1-naphthyl isocyanate and 1-naphthylamine formation. It is suggested that 1-naphthyl isocyanate, 1-naphthylamine and the highly reactive sulfur released from 1-naphthylisothiocyanate might be involved in the hepatotoxic effect of 1-naphthylisothiocyanate.     

The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1)

Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. KD values of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.     

Inhibition of procarcinogen-bioactivating human CYP1A1, CYP1A2 and CYP1B1 enzymes by melatonin

Abstract:  Administration of melatonin to rodents decreases the incidence of tumorigenesis initiated by benzo[ a ]pyrene or 7,12-dimethylbenz[ a ]anthracene, which requires bioactivation by cytochrome P450 enzymes, such as CYP1A1, CYP1A2 and CYP1B1, to produce carcinogenic metabolites. The present study tested the hypothesis that melatonin is a modulator of human CYP1 catalytic activity and gene expression. As a comparison, we also investigated the effect of melatonin on the catalytic activity of CYP2A6, which is also a procarcinogen-bioactivating enzyme. Melatonin (3–300 μ m ) decreased 7-ethoxyresorufin O -dealkylation catalyzed by human hepatic microsomes and recombinant CYP1A1, CYP1A2 and CYP1B1, whereas it did not affect coumarin 7-hydroxylation catalyzed by hepatic microsomes or recombinant CYP2A6. Melatonin inhibited CYP1 enzymes by mixed inhibition, with apparent K _i values (mean ± S.E.M.) of 59 ± 1 (CYP1A1), 12 ± 1 (CYP1A2), 14 ± 2 (CYP1B1) and 46 ± 8 μ m (hepatic microsomes). Additional experiments indicated that melatonin decreased benzo[ a ]pyrene hydroxylation catalyzed by hepatic microsomes and CYP1A2 but not by CYP1A1 or CYP1B1. Treatment of MCF-10A human mammary epithelial cells with melatonin (up to 300 μ m ) did not affect basal or benzo[ a ]pyrene-inducible CYP1A1 or CYP1B1 gene expression. Consistent with this finding, melatonin did not influence reporter activity in aryl hydrocarbon receptor-dependent pGudluc6.1-transfected MCF-10A cells treated with or without benzo[ a ]pyrene, as assessed in an in vitro cell-based luciferase reporter gene assay. Overall, melatonin is an in vitro inhibitor of human CYP1 catalytic activity, and it may be useful to develop potent analogues of melatonin as potential cancer chemopreventive agents that block CYP1-mediated chemical carcinogenesis.     

甲型H1N1与季节性H1N1流感病毒血凝素基因比较分析

目的分析泰安市2008～2009年度季节性流感与2009年度甲型H1N1流感病原学检测结果 ,比较季节性H1N1与甲型H1N1血凝素基因变异情况。方法选择国家级流感监测哨点医院以及暴发疫情的疫点,采集流感样病例的鼻咽拭子标本,通过RealtimePCR进行病毒检测,用MDCK细胞进行病毒分离,通过RT-PCR扩增血凝素HA1片段的基因并测序,利用生物信息学进行序列分析。结果 2008～2009年共检测鼻咽拭子标本283份,分离出流感病毒33株,分离阳性率为11.67%,其中季节性H1N1亚型31株。2009年5月1日～12月31日,检测鼻咽拭子标本996份,流感核酸检测阳性417份,阳性率为41.86%,其中甲型H1N1337份,季节性H1N1亚型1份。6株季节性H1N1病毒均在多个氨基酸位点上发生变异,与疫苗株A/Brisbane/59/2007(H1N1)比较,有11个位点发生了突变,其中5个位点位于抗原决定簇上;测序成功的6株甲型H1N1病毒在多个氨基酸位点发生变异,与疫苗株A/California/07/2009(H1N1)比较,有6个位点发生突变,其中1个位点位于抗原决定簇的B区。结论 2008～2009年度季节性H1N1为优势株,甲流暴发后,甲型H1N1成为绝对优势毒株。季节性H1N1分离株有多处氨基酸替换,抗原决定簇B区变异频繁;甲型H1N1病毒分离株的基因有变异,但关键位点第222位仍为D(天冬氨酸),与疫苗株相比抗原决定簇的关键位点变化不大。     

Characterization of MTHFR,GSTM1, GSTT1, GSTP1, and CYP1A1 genotypes in childhood acute leukemia

The role of methylenetetrahydrofolate reductase (MTHFR C677T), glutathione S-transferases (GSTM1 and GSTT1 null, GSTP1 Ile105Val), and cytochromes p450 (CYP1A1*2A) genotypes in the etiology of childhood leukemia was simultaneously investigated. 144 Turkish children with acute lymphoblastic leukemia (ALL) and 33 with acute nonlymphoblastic leukemia (ANLL) were studied and compared with 185 healthy pediatric controls. The frequency of MTHFR genotype was insignificantly higher in ALL (7.7%) and ANLL (6.3%) than in controls (4.4%). Equal distribution of the GSTM1 null genotype was detected between ALL patients and controls (55%), while its incidence was slightly higher in ANLL patients (61.3%). Although GSTT1 null genotype was insignificantly lower in ALL patients (20.9%) than controls (22.7%), it was significantly underrepresented in ANLL patients (6.5%) (P = 0.05, OR 0.24, 95% CI 0.05-1.03). The homozygous frequency of GSTP1 genotype did not differ significantly between groups of ALL (3.7%), ANLL patients (9.1%) and controls (4.9%). Homozygous CYP1A1*2A genotype was underrepresented in ALL patients (1%) as compared to control (4.8%) but the differences did not reach to statistical significance (OR 0.21; 95% CI 0.03-1.72). Homozygosity for this genotype was not detected in ANLL patients. No particular association was noted between different combinations of combined genotypes and risk of development of childhood ALL and ANLL. These results suggested that there are no significant associations between the studied genotypes and the risk of developing either form of acute leukemia except GSTT1 null and homozygosity for CYP1A1 genotypes that may play protective roles in the development of ANLL in Turkish children.