BrdU免疫荧光染色方法的改进

目的探索一种能确保BrdU与其他标志物免疫荧光染色顺利进行的方法。方法本实验采用三种不同的过酸方法来进行BrdU免疫荧光染色:即①传统染色法;②过酸提前法;③室温过酸法。采集图像,观察三种方法的实验结果并统计实验数据。结果①传统染色法:BrdU阳性标记定位明确,与之双标的标志物DCX亦可正常显色,但核酸染料bisbenzimide却未能正常显色;②染色提前法:BrdU与DCX均可正常显色,而bisbenzimide显色不稳定;③室温过酸法:BrdU与DCX、bisbenzimide均能正常显色,且荧光亮度适中,效果理想。结论室温过酸法能确保BrdU与其他标志物双标正常显色,同时又能保证用来明确BrdU处理过的活细胞增殖状态的bisbenzimide染料正常显色,是目前一种较好的能够辨别BrdU假阳性的免疫荧光染色方法。     

丰富环境对血管性痴呆模型大鼠梨状皮质未成熟神经元表达的影响

BACKGROUND:Researches showed that the enriched environment could improve the cognitive dysfunction of rats with vascular dementia. However, there are few reports regarding its mechanism of action. OBJECTIVE:To explore the effect of enriched environment on the cognitive dysfunction of rats with vascular dementia from the behavioral level.  METHODS:Vascular dementia models were made by permanent ligation of bilateral common carotid arteries and were divided into vascular dementia group (n=8) and enriched environment group (n=12). Vascular dementia group was taken care under conventional breeding environment for 30 days, while the enriched environment group was subjected to the enriched environment for 30 days. Morris water maze test was adapted to test the cognitive function of rats between two groups. Immunohistochemistry and immunoblotting were applied to observe the number of DCX⁺ cells and DCX protein level in both groups. The number of DCX^-labeled cells co-expressing NeuN was observed using immunofluorescence technique. RESULTS AND CONCLUSION:(1) The escape latency in the vascular dementia group was longer than that in the enriched environment group (P < 0.05). The times across the platform was less in the vascular dementia group than that in the enriched environment group (P < 0.05). (2) In comparison with the enriched environment group, the number of DCX-positive cells and its protein level in the piriform cortex were significantly decreased in the vascular dementia group (P < 0.05). (3) The number of DCX/NeuN co-labeled cells in the piriform cortex was significantly less in the vascular dementia group than in the enriched environment group (P < 0.05). (4) These findings suggested that enriched environment could improve the cognitive dysfunction of rats with vascular dementia through promoting the expression and differentiation of the immature neurons.     

不同浓度十二烷基磺酸钠在提取甲醛固定石蜡包埋组织β-淀粉样前体蛋白剪切酶-1中的作用

目的:探索从甲醛固定、石蜡包埋(formaldehyde-fixed,paraffin-embedded,FFPE)的脑组织中提取β-淀粉样前体蛋白剪切酶-1(BACE-1)的有效方法.方法:将大鼠 FFPE 脑组织分别加入不同浓度十二烷基磺酸钠(SDS,0%,0.5%,1%,2%和4%)提取 BACE-1,新鲜大鼠腩组织作为对照组.提取的 BACE-1 蛋白量采用 Western blot 进行检测.结果:未用SDS 处理的同定组织提取不到 BACE-1,随着 SDS 浓度的逐渐升高,同定组织中提取的 BACE-1 蛋白也逐渐增加,4%SDS处理的 FFPE 组织与新鲜组织提取的 BACE-1 蛋白量没有明显差别.结论:SDS 处理是 FFPE 组织提取 BACE-1 的必需条件,高浓度(4%)SDS 处理是 FFPE 组织提取 BACE-1 的一种有效方法.     

阿尔茨海默病老年斑相关研究进展

正阿尔茨海默病(Alzheimer's disease,AD)是一种以进行性痴呆为特征的年龄相关性神经退行性疾病,老年斑(senile plaques,SP)为其典型病理特征之一~([1-2])。随着世界范围内老龄化社会的到来,AD发病率呈逐年递增趋势。来自世界卫生组织的报道称,目前全世界范围内该病患者高达3 560万,每20年患病人数将翻一番,预计2050年可达到1.154亿人~([3]),这一疾病正在成为国际神经疾病研究的热点。1907年,德国神经病理学     

三七总皂苷对全脑缺血大鼠海马脑水肿及GFAP表达的影响

目的探讨三七总皂苷(TSPN)对全脑缺血后成年大鼠海马胶质纤维酸性蛋白(GFAP)表达及脑水肿的影响。方法采用四血管阻断法制作全脑缺血模型,大鼠被随机分成假手术组、模型组和TSPN组。TSPN组大鼠全脑缺血后30 min ip 75 mg/kg TSPN,每天1次,假手术组和模型组ip给予等体积的生理盐水,连续14 d。分别于再灌注1、3、7、14 d后处死。采用干湿法计算各组大鼠脑组织含水量,免疫组织化学染色观察海马齿状回(SGZ)区GFAP的表达,免疫荧光双标观察SGZ区GFAP/DCX的共表达情况,免疫印迹技术检测海马CA1区GFAP蛋白表达水平。结果 TSPN组大鼠脑组织含水量显著低于模型组(P0.05);TSPN组、模型组SGZ区GFAP+细胞密度值在3、7、14 d有统计学差异(P0.05);两组SGZ区GFAP/DCX共表达细胞与DCX阳性表达细胞数目的比值在第7、14天差异显著(P0.05);免疫印迹结果显示TSPN组海马CA1区GFAP蛋白表达水平在第3、7、14天显著高于模型组(P0.05)。结论 TSPN可通过促进全脑缺血后大鼠SGZ区星形胶质细胞活化、促进神经再生、减轻脑水肿而发挥神经保护作用。     

1000/铅暴露早期诱导成年大鼠视网膜神经元BACE-1表达上调

Previous studies have reported that non-human primates and rodents exposed to lead during brain development may become dependent on the deposition of pre-determined β-amyloid protein (Aβ),and exhibit upregulation of β-site amyloid precursor protein expression in old age.However,further evidence is required to elucidate the precise relationship and molecular mechanisms underlying the effects of early lead exposure on excessive Aβ production in adult mammals.The present study investigated the effects of lead exposure on expression of β-amyloid precursor protein cleavage enzyme-1 (BACE-1) in the rat retina and the production of Aβ in early development,using the retina as a window for studying Alzheimer’s disease.Adult rats were intraocularly injected with different doses of lead acetate (10μmol/L,100μmol/L,1 mmol/L,10 mmol/L and 100 mmol/L).The results revealed that retinal lead concentration,BACE-1 and its cleavage products β-C-terminal fragment and retina Aβ1-40 were all significantly increased in almost all of the lead exposure groups 48 hours later in a dose-dependent manner.The only exception was the 10μmol/L group.The distribution of BACE-1 in the retina did not exhibit obvious changes,and no distinctive increase in the activation of retinal microglia was apparent.Similarly,retinal synaptophysin expression did not exhibit any clear changes.These data suggest that lead exposure can result in the upregulation of retinal neuron BACE-1 expression in the early period of development and further increase the overproduction of Aβ1-40 in the retina.Our results provided novel insight into the molecular mechanisms underlying environmentally-induced Alzheimer’s disease.     

人胎脊髓胸段一氧化氮合酶阳性神经元发育的研究

人胎脊髓胸段一氧化氮合酶阳性神经元发育的研究神经生物学研究室雷德亮，卢大华，刘丹，周丙林，陈二云，严小新，罗学港，刘忠浩提要取１２例妊娠１５１３６周胎儿脊髓胸段标本，用还原型辅酶Ⅱ组化方法观察含一氧化氨合酶（ＮＯＳ）神经元的发育，并用体视学方法对其数...     

Sp8在健康大鼠侧脑室室管膜区的表达

目的:探讨健康大鼠侧脑室室管膜区(SVZ区)Sp8与微管相关蛋白Doublecortin(DCX)的表达。方法:1、6、12月龄SD大鼠被处死。脑冠状位切片,免疫荧光单标染色观察1、6、12月龄组SVZ区Sp8细胞与DCX细胞的表达,免疫荧光双标染色观察1月龄组Sp8/GFAP、Sp8/Ki67的共表达情况。结果:1月龄组SVZ区Sp8细胞、DCX细胞密度均显著高于6月龄组及12月龄组;除此之外,1月龄组SVZ区有Sp8/GFAP、Sp8/Ki67的共表达细胞。结论:Sp8与DCX在大鼠的SVZ区有广泛的表达;Sp8调控SVZ区神经发生和星形胶质干细胞的分化。     

博士研究生神经解剖学课程研讨式教学模式的探索

正博士研究生是国家培养人才的最高层次。博士研究生经过本科、硕士阶段的学习,已经掌握专业领域的基础知识和研究方法,博士研究生培养的重要特征是开发其独立、自主、有效地开展和探索科学研究的能力。创新性是博士学位论文的灵魂,也是衡量博士学位论文质量水平的重要标志~([1-2])。中南大学湘雅医学院博士研究生开设的神经解剖学课程(选修课),旨在开发