【作者】 谷峰；

【导师】 罗焕敏；

【作者基本信息】 暨南大学 ， 药理学， 2003， 硕士

【摘要】 目的：通过体外孵育的方法研究β淀粉样蛋白42(Aβ₄₂)聚集、聚集后对体外培养海马神经元的毒性作用，观察牛膝醇提物（Nx-E）抑制Aβ₄₂聚集和减小其神经毒性的作用。 方法：（1）Aβ₄₂单独或与低（1.25％）、中（2.5％）、高（5％）剂量Nx-E共同在37℃，5％CO₂培养箱中孵育后，使用硫磺素T（Th-T）荧光分析和透射电镜（TEM）观察；（2）体外培养SD新生大鼠海马神经元，采用NSE免疫组化染色法进行神经元鉴定；（3）在培养液中加入Aβ₄₂单独或与低、中、高Nx-E共同孵育的产物，进行形态学观察、定量分析、四唑盐（MTT）比色实验和乳酸脱氢酶（LDH）渗漏量测定，分析各组孵育产物的神经细胞毒性大小。 结果：（1）随着孵育时间延长，Aβ₄₂的荧光强度逐渐增加，尤其7d时荧光强度明显升高（P＜0.01），TEM观察以淀粉样纤维结构为主；（2）2.5％和5.0％的Nx-E使Aβ₄₂的荧光强度减小，TEM观察5.0％Nx-E和Aβ₄₂孵育7d后，淀粉样纤维明显减少；（3）染成棕褐色的细胞占大多数，表明所培养细胞大多数为神经元；（4）终浓度25μmol·L^-1的Aβ₄₂使海马神经元细胞数、突起数目和长度减小，为Aβ₄₂的毒性剂量；（5）Aβ₄₂和2.5％、5.0％Nx-E共同孵育的产物使海马神经元细胞数、突起数目和长度增加，四唑盐（MTT）比色实验吸光度增加，LDH活力减小。 结论：0.25mmol·L^-1Aβ₄₂孵育7d发生明显聚集，形成了大量的淀粉样纤维；Aβ₄₂孵育7d对海马神经元的毒性浓度为25μmol·L^-1；2.5％和5％的Nx-E能抑制Aβ₄₂聚集和淀粉样纤维的形成，减小Aβ₄₂的神经毒性，其机制可能和抗氧化作用有关。更多还原

【Abstract】 Objectives: To study 3 -amyloid protein 42 (Aβ₄₂ aggregation and amyloid fibril formation induced by incubation in vitro, and observe the neurotoxictity of Aβ₄₂ to rat hippocampal neurons. To observe the effects of ethanol extracts of Achyranthes bidentata blume （Nx-E） inhibiting A 3 42 aggregation and reducing the neurotoxicittOf (Aβ₄₂.Methods: （1）(Aβ₄₂ alone and (Aβ₄₂ co-incubated with 1.25%, 2.5% or 5% Nx-E solutions were incubated in 37 , 5% CO2 incubator, then were monitored by thioflavine-T fluorescence and observed by transmission electron microscopy （TEM）; （2） The newborn Sprague-Dawley rat hippocampal neurons were cultured and identified by NSE stain; （3） After incubation, the solutions of Aβ₄₂ or Aβ₄₂ co-incubated with 1.25%, 2.5%, 5.0% Nx-E were added to cell cultured medium.The numbers & length of neurites were measured with the convert phase microscope, and MTT & lactate dehydrogenase （LDH） assay were done to analyze the toxicity to hippocampal neurons.Results: （1） Thioflavine-T fluorometr y results showed that the fluorescence intensity increased with the time of Aβ₄₂ incubation going. The fluorescence intensity of (Aβ₄₂ non-incubated was near to that of the backgroud, and at the 7th day 0.25mmol L -1 Aβ₄₂ fluorescence intensity was significantly increased （P<0.01）. Little amyloid fibril formed in non-incubated group but much did at the 7th day; （2） The fluorescence intensity in 2.5% Nx-E group reduced, and in 5.0% reduced remarkablely. Little fibril but many amorphous materials were observed; （3） Most of the newborn rat hippocampal cells became brown, suggesting them being neurons; （4） 25 mol L-1 Aβ₄₂ could reduce more numbers and the length of neurites than that of 12.5 mol L-1, so the former was used as the celltoxity concentration; （5） Compared with 25 mol L-1 Aβ₄₂, the solution of A 3 42 co-incubated with 2.5% or5.0% Nx-E could increase numbers and the length of neurites. The optical density（OD） of MTT assay increased and LDH decreased.Conclusions: Incubated up to 7 days alone, Aβ₄₂ aggregates and forms amyloid fibril.The neurotoxitityconcentration of Aβ₄₂ is 25 mol L-1. 2.5 % and 5 %Nx-E canprevent Aβ₄₂ from polymerizing and forming fibril, and reduce the neurotoxitity Themechanism may be Nx-E has anti-oxidative activity.更多还原