Colivelin拮抗淀粉样β蛋白所致大鼠记忆损伤的神经保护作用研究

发布时间：2018-05-12 21:39

本文选题：Colivelin + β淀粉样蛋白　；参考：《山西医科大学》2012年硕士论文

【摘要】：阿尔茨海默病（Alzheimer’s disease, AD）是一种原发性中枢神经系统退行性疾病，其主要临床表现为进行性认知功能障碍、学习和记忆能力丧失、智力下降，晚期出现严重痴呆。目前，全世界约有3600万AD患者，据ADI估计这个数字以每20年翻一倍的速度增长,到2030年将会达到6600万人。我国AD患者目前超过500万，并且随着人口老龄化速度和程度的加剧，我国AD的发病率和患者总数还将持续增加。毫无疑问，AD已成为21世纪威胁人类的最严重疾病之一，是继心血管疾病、脑血管疾病和癌症之后严重影响老年人身体健康和人类寿命的“第四大杀手”。 AD典型病理特征之一是脑内出现高密度的老年斑。研究表明，老年斑的主要成分是由39-43个氨基酸组成的淀粉样β蛋白（Amyloidβ-protein，Aβ）。全长Aβ片段的神经毒性作用已有广泛报道，在此基础之上，有大量实验表明Aβ25-35是全长Aβ分子中一个较短的活性片段。本研究室在以往的研究中也发现，Aβ25-35能在细胞培养、离体膜片钳和在体电生理实验中表现出与全长Aβ类似的神经毒性作用。这些研究提示，拮抗Aβ神经毒性作用可能是治疗AD的一个有效手段。然而，迄今为止针对Aβ的AD治疗尤其是有效拮抗Aβ神经毒性作用的药物研究，仍然没有突破性进展。 Humanin（HN）是日本学者于2001年首次发现的一个由24个氨基酸组成的线性多肽，能够特异性抑制AD及其各种相关损伤引起的神经元细胞死亡。Colivelin是由日本学者于2005年首次报道的一种新的、由26个氨基酸组成的HN衍生物。目前有部分研究显示，Colivelin有很强的神经保护作用，能够有效逆转Aβ在原代培养皮层神经元和行为学实验中表现出的神经毒性。然而，对于Colivelin在整体动物中的作用，尤其是对学习记忆的电生理模型长时程增强（long-term potentiation, LTP）的影响及Colivelin保护作用的机制的研究还处于起步阶段。 Morris水迷宫（Morris water maze, MWM）是用来衡量动物学习记忆功能的经典的行为学实验手段，能够有效反映动物的认知功能改变，因而被广泛应用于动物学习记忆功能包括AD动物行为学测试研究。长时程增强(long term potentiation, LTP)是由高频刺激(high-frequency stimulus, HFS)所引发的一种兴奋性突触传递效能长时间持续增强的现象。鉴于海马是AD发病过程中最早和最易受累的脑区之一，且海马LTP的伤害与动物认知缺损高度相关，海马LTP特别是在体LTP已被视为研究学习记忆和AD发病机制的一个重要的电生理细胞模型而受到普遍关注。因此，本实验采用海马内分别给予Aβ25-35、生理盐水、Colivelin（0.2 nmol）和Colivelin（0.2 pmol、2 pmol、0.2 nmol）+Aβ25-35的方法，观察了Colivelin对大鼠学习记忆的影响，并探讨其对于Aβ25-35导致的学习记忆功能损伤的逆转作用。实验研究分为以下两部分：（1）利用国际公认的经典的Morris水迷宫测试手段，观察Colivelin预处理是否能有效拮抗大鼠Aβ所致的学习记忆伤害；（2）利用在体海马LTP记录手段，观察海马内注射Colivelin预处理对于Aβ25-35引起的LTP压抑的作用，进一步阐明Colivelin神经保护作用的突触前、后机制。通过以上两部分研究，为Colivelin参与神经系统功能调节提供充分的实验证据，从而为保护神经元免受Aβ神经毒性伤害，为预防和治疗AD提供新的思路和理论依据。第一部分：Colivelin部分逆转Aβ25-35引起的大鼠空间学习记忆功能损害目的：利用Morris水迷宫测定大鼠空间学习记忆功能，观察海马内给予Aβ25-35、生理盐水、Colivelin以及合用Colivelin和Aβ25-35后大鼠在Morris水迷宫中的行为学表现，探讨Colivelin预处理对Aβ引起的空间学习记忆功能损伤可能的作用。方法：取运动灵活、无视力障碍的成年雄性SD大鼠(200-230g)随机分组，进行Morris水迷宫定位航行和空间探索实验，分别测定大鼠的空间学习和记忆能力。主要指标包括大鼠寻找平台的平均逃避潜伏期和游泳距离、撤离平台后大鼠在目标象限游泳时间（限定时间为：120秒）和距离。同时，测定各组大鼠的游泳速度及大鼠的视力，以排除运动能力与视力障碍对测定指标的影响。各种药物均经海马内注射给予，恢复2-3周后进行实验。结果：（1）对照组大鼠学习记忆功能正常。逃避潜伏期在训练第1-5天内分别为79.0±3.3 s, 36.6±3.0 s, 20.2±2.0 s, 19.9±1.3 s和16.5±1.7 s；大鼠找到平台所游过的距离分别为1427.5±135.1 cm, 689.4±80.8 cm, 369.2±50.7 cm, 322.4±40.6 cm和265.3±44.0 cm；撤除平台后大鼠在目标象限游泳所用时间和距离分别为55.4±3.0s和1150.4±63.9cm。（2）海马内注射4nmol Aβ25-35后显著降低了大鼠的空间学习和记忆能力。Aβ25-35海马内注射组大鼠的平均潜伏期和游过距离在第1天与对照组没有明显差别，在第2-5天分别为52.8±4.3 s, 39.5±1.6 s, 29.3±2.6 s, 26.8±1.4 s和899.1±108.9 cm, 551.7±54.9 cm,448.5±68.7 cm, 369.6±28.6 cm，与对照组相比，其平均潜伏期明显延长，游过距离明显缩短（P0.01或P0.05）；撤除平台后大鼠在目标象限游泳所占时间和距离显著缩短，分别为36.8±2.9 s和756.9±52.9 cm（P0.01）。（3）海马内单独注射0.2 nmol Colivelin后，大鼠的学习、记忆功能与对照组相比均未受到影响（P0.01），分别为35.3±4.4 s,22.1±2.9 s, 21.4±2.6 s, 19.4±0.7 s和670.0±90.6 cm, 398.3±80.9 cm, 357.2±51.5 cm,260.0±37.0 cm，撤掉平台后在目标象限游泳所用的时间和距离为55.0±3.0 s和1148±63.1cm，与对照组相比无明显差别（P0.01）。但联合给予Colivelin（0.2 pmol,2 pmol,0.2nmol）与Aβ25-35后，与Aβ25-35组相比，各组平均潜伏期和游过距离两个指标在第2-5天都有缩短现象，0.2 pmol Colivelin+ Aβ25-35组(n=11)2-5天找到平台所用的时间和距离分别为45.4±3.5 s, 33.9±1.7 s, 24.6±3.8 s, 22.3±1.9 s和728.0±78.3 cm, 425.1±43.8 cm,339.9±84.4 cm, 286.1±41.5 cm；在2 pmol Colivelin+ Aβ25-35组(n=12)分别为33.8±3.8 s,29.8±2.1 s, 22.0±1.6 s, 20.0±1.6 s和582.1±115.2 cm, 385.6±59.1 cm, 281.5±33.3 cm,228.8±27.3 cm；在0.2 nmol Colivelin+ Aβ25-35组(n=12),分别为28.1±2.4 s, 20.8±2.1 s,19.3±1.6 s, 17.9±2.4 s和509.5±63.6 cm,302.9±56.7 cm, 247.9±28.8 cm, 203.8±47.9 cm与Aβ25-35组相比均有明显缩短（P0.01），并且这种逆转作用具有剂量依赖性。撤除平台后大鼠在目标象限游泳所用时间和距离实验中，0.2 pmol Colivelin+ Aβ25-35组为39.1±2.1s和799.0±74.0 cm；2 pmol Colivelin+ Aβ25-35组为43.8±3.0 s和934.9±53.8 cm；0.2nmol Colivelin+ Aβ25-35组为54.0±3.7 s和1148.4±63.0 cm，与Aβ25-35组相比均有并不同程度的增加，并且这种增加呈现出剂量依赖性。（4）各实验组动物在可视平台实验中的逃避潜伏期和游泳速度均无显著区别。结论：海马内注射Aβ25-35能够伤害大鼠空间学习和记忆能力，单独使用Colivelin不会对大鼠的学习和记忆活动造成影响，但Colivelin预处理之后给予Aβ25-35，，发现Colivelin可部分逆转Aβ对大鼠空间学习记忆功能的损伤作用，并且此逆转作用具有剂量依赖性。第二部分: Colivelin拮抗Aβ25-35引起的大鼠在体海马LTP损害目的：通过海马内注射Colivelin以及Aβ25-35后记录海马场兴奋性突触后电位（field excitatory postsynaptic potentials, fEPSPs）和长时程增强（LTP），研究不同剂量Colivelin预处理之后对Aβ25-35压抑大鼠海马CA1区在体LTP的逆转作用及其可能机制。方法：采用海马内注射给药后已行水迷宫测试的SD大鼠，麻醉后将其固定在脑立体定位仪上，将绑定好的刺激电极和记录电极精确插入到海马刺激和记录部位。通过给予海马Schaffer侧枝单个电刺激、强直电刺激和双脉冲刺激，在海马CA1区放射层诱发和记录基础的fEPSPs、强直刺激引起的LTP，以及配对脉冲引起的双脉冲易化（pairedpulse facilitation, PPF）。结果：（1）Aβ25-35不影响基础性突触传递，但对于高频刺激引起的LTP具有明显的压抑作用。给予HFS后0 min,30 min,60 min，对照组fEPSPs幅度增强至176.8±4.1%,150.6±2.8%和141.3±2.3%；4 nmol Aβ25-35组的fEPSPs幅度分别只有166.6±3.0%,116.2±1.6和106.2±2.8%；表明Aβ25-35片段可明显压抑海马CA1区LTP (P0.01)（2）单独给予0.2 nmol Colivelin后，基础性突触传递和LTP都没有发生明显改变。其在给予HFS后0 min,30 min,60 min时分别为：178.5±2.9%，139.9±2.9%，138.5±2.1%。给予Colivelin(0.2 pmol,2 pmol,0.2 nmol)预处理后，再给予Aβ25-35，在0.2 pmol Colivelin+Aβ25-35组，其数值分为为：164.5±1.8%，122.4±1.9%，110.3±1.1%；在2 pmolColivelin+ Aβ25-35组中，数值分别为：166.0±2.3%，132.2±2.1%，119.2±1.2%；在0.2nmol Colivelin+ Aβ25-35组中则分别为：178.7±2.8%，139.8±2.4%，126.0±2.6%。LTP值在HFS后0 min, 30 min和60 min时的数值均高于单独给予Aβ25-35 LTP (P0.01)，并且差别具有剂量依赖性。表明Colivelin能够剂量依赖性的逆转Aβ对LTP的伤害作用。（3）对照组和各实验组的PPF都没有显著差异，提示所用各种药物影响LTP主要不是通过突触前机制实现的。结论：海马内注射Aβ25-35能够压抑大鼠海马在体LTP；单独使用Colivelin不会对海马的基础性传递功能及LTP造成影响，但是先于Aβ25-35预处理可以部分改善Aβ25-35对LTP的压抑作用，并且这种作用具有剂量依赖性。总之，本研究分别利用Morris水迷宫和LTP两种实验方法，证明了Colivelin可有效对抗Aβ所致的行为学和海马LTP的伤害。因此，本研究为AD的预防和治疗提供了一个新的线索和思路。
[Abstract]:Alzheimer 's disease (AD) is a primary neurodegenerative disease of the central nervous system. Its main clinical manifestations are progressive cognitive impairment, loss of learning and memory, loss of intelligence, and advanced dementia. At present, about 36 million AD patients worldwide, according to ADI, have doubled the number to double every 20 years. The rate of growth will reach 66 million in 2030. The AD patients in China are now more than 5 million, and with the increasing speed and degree of population aging, the incidence of AD and the total number of patients in China will continue to increase. There is no doubt that AD has become one of the most serious diseases that threaten human beings in twenty-first Century, which is followed by cardiovascular disease, cerebrovascular disease and After cancer, the "fourth killer" seriously affects the health and life span of the elderly.
One of the typical pathological features of AD is the high density of the senile plaques in the brain. Studies have shown that the main component of the senile plaques is Amyloid beta -protein (A beta), which consists of 39-43 amino acids. The neurotoxicity of the full length A beta fragment has been widely reported. On this basis, a large number of experiments have shown that A beta 25-35 is the full length of the A beta molecule. A shorter active fragment. In previous studies, this study also found that A beta 25-35 showed neurotoxicity similar to full-length A beta in cell culture, in vitro patch clamp and in body electrophysiological experiments. These studies suggest that antagonism to the neurotoxicity of A beta may be an effective means for the treatment of AD. There is still no breakthrough in the study of A beta AD therapy, especially for the effective anti A beta neurotoxicity.
Humanin (HN) is a linear polypeptide composed of 24 amino acids for the first time in 2001 by Japanese scholars. It is a new type of HN derivative, composed of 26 amino acids, which was first reported by Japanese scholars in 2005 by Japanese scholars in 2005. It is a new kind of HN derivative, which is first reported by Japanese scholars in 2005. It shows that Colivelin has a strong neuroprotective effect and can effectively reverse the neurotoxicity of A beta in primary cultured cortical neurons and behavioural experiments. However, the effect of Colivelin on the whole animal, especially the effect of long-term potentiation (LTP) on the electrophysiological model of learning and memory, and Colivel The mechanism of in protection is still in its infancy.
The Morris water maze (Morris water maze (MWM)) is a classic behavioral experiment used to measure the learning and memory function of animals. It can effectively reflect the changes in cognitive function of animals. Therefore, it is widely used in animal learning and memory function, including the study of AD animal behavior test. Long term enhancement (long term potentiation, LTP) is a high frequency (long term potentiation, LTP). High-frequency stimulus (HFS) triggering a prolonged and continuous enhancement of the efficiency of excitatory synaptic transmission. Given that the hippocampus is one of the earliest and most vulnerable brain regions in the AD pathogenesis, and the damage of the hippocampus LTP is highly related to the cognitive impairment of animals, and the hippocampus LTP, especially in the body LTP, has been considered to study learning memory and AD. The pathogenesis of an important electrophysiological cell model has attracted widespread attention.
Therefore, in this experiment, A beta 25-35, saline, Colivelin (0.2 nmol) and Colivelin (0.2 pmol, 2 pmol, 0.2 nmol) +A beta 25-35 were used to observe the effect of Colivelin on learning and memory in rats and to explore the reversal effect of A beta 25-35 on the impairment of learning and memory function. The experimental study was divided into two parts. (1) using the internationally recognized classic Morris water maze test to observe whether Colivelin preconditioning can effectively antagonize the learning and memory injury caused by A beta in rats; (2) to observe the effect of Colivelin preconditioning on A beta 25-35 induced by A beta 25-35 in hippocampal LTP recording, and to further clarify Colivelin neuroprotection. Through the above two parts, we provide sufficient experimental evidence for the involvement of Colivelin in the function regulation of the nervous system, thus providing a new idea and theoretical basis for the protection of neurons from A beta neurotoxicity and for the prevention and treatment of AD.
Part one: Colivelin partially reverses the impairment of spatial learning and memory induced by A beta 25-35 in rats.
Objective: to determine the spatial learning and memory function of rats by Morris water maze, and observe the behavior of A beta 25-35, saline, Colivelin, and Colivelin and A beta 25-35 rats in the Morris water maze, and explore the possible effect of Colivelin preconditioning on the impairment of spatial learning and memory impairment induced by A beta.
Methods: the adult male SD rats (200-230g) without visual impairment were randomly divided into the Morris water maze navigation and space exploration experiment. The spatial learning and memory ability of the rats were measured respectively. The main indexes included the average escape latency and swimming distance of the rats in search of the platform. The rats were in the target image after the evacuation platform. Swimming time limited (120 seconds) and distance were limited. At the same time, the swimming speed of rats and the visual acuity of rats were measured in order to exclude the influence of motor ability and visual impairment on the measurement index. All kinds of drugs were given by intramuscular injection in the hippocampus, and the experiment was carried out after 2-3 weeks.
Results: (1) the learning and memory function of the control group was normal. The escape latency was 79 + 3.3 s, 36.6 + 3 s, 20.2 + 2 s, 19.9 + 1.3 s and 16.5 + 1.7 s, respectively. The time and distance of the rats in the target quadrant swimming were 55.4 + 3.0s and 1150.4 + 63.9cm. (2) intramuscular injection of 4nmol A beta 25-35. The average latency and distance of the rats in the group of intramuscular injection of.A beta 25-35 were not significantly different from those in the control group at the 2-5 day on the 2-5 day. They were 52.8 + 4.3 s, 39.5 + 1.6 s, 29.3 + 2.6 s, 26.8 + 1.4 s and 899.1 + 108.9 cm, 551.7 + 54.9 cm, 448.5 + 39.5 cm. Compared with the control group, the average latency period was obviously prolonged and the travel distance was obviously shortened (P0.01 or P0.05). After the removal of the platform, the rats in the target quadrant swimming time and distance were significantly shortened, respectively. For 36.8 + 2.9 s and 756.9 + 52.9 cm (P0.01). (3) a single injection of 0.2 nmol Colivelin in the hippocampus, the learning and memory function of the rats were not affected by the control group (P0.01), respectively 35.3 + 4.4 s, 22.1 + 2.9 s, 21.4 + 2.6 s, 19.4 + s and cm cm. The time and distance of the target quadrant swimming were 55 + 3 s and 1148 + 63.1cm, and there was no significant difference compared with the control group (P0.01). However, compared with the A beta 25-35 group with Colivelin (0.2 pmol, 2 pmol, 0.2nmol) and A beta 25-35, the average latency and the travel distance two indexes were shortened on the 2-5 day, 0.2 pmol Colivelin+ A. The time and distance to find the platform for the 2-5 days of group n=11 (n=11) were 45.4 + 3.5 s, 33.9 + 1.7 s, 24.6 + 3.8 s, 22.3 + 1.9 s and 728 + 78.3 cm, 425.1 + cm and cm, respectively. 385.6 + 59.1 cm, 281.5 + 33.3 cm, 228.8 + 27.3 cm, 0.2 nmol Colivelin+ A beta 25-35 (n=12), 28.1 + 2.4 s, 20.8 + 2.1 s and 19.3 + s respectively. After the removal of the platform, the 0.2 pmol Colivelin+ A beta 25-35 groups were 39.1 + 2.1S and 799 + 74 cm, and 2 pmol Colivelin+ A beta 25-35 groups were 43.8 + 3 s and 934.9 + 25-35 cm. The increase in different degrees and this increase showed a dose dependence. (4) there was no significant difference between the escape latency and the swimming speed of the experimental groups in the visual platform experiment.
Conclusion: intramuscular injection of A beta 25-35 can harm the spatial learning and memory ability of rats. The use of Colivelin alone can not affect the learning and memory activities of rats, but A beta 25-35 is given after Colivelin preconditioning. It is found that Colivelin can partly reverse the damage effect of A beta on the spatial learning and memory function of rats, and this reversal effect is also found. There is a dose-dependent manner.
The second part: Colivelin antagonize the LTP damage induced by A beta 25-35 in the hippocampus of rats.
Objective: to record the excitatory postsynaptic potential (field excitatory postsynaptic potentials, fEPSPs) and long term potentiation (LTP) in hippocampal field after injection of Colivelin and A beta 25-35 in the hippocampus, and to study the reversal effect and possible mechanism of the CA1 region of hippocampus in the CA1 region of A beta 25-35 in rats after different doses of Colivelin preconditioning.
Methods: SD rats were tested with water maze test after intralanhippocampal injection. After anesthesia, they were fixed on the stereotactic brain locator. The binding stimulation electrode and recording electrode were inserted into the hippocampus stimulation and recording site accurately. By giving a single electrical stimulation to the hippocampal Schaffer side branch, direct electrical stimulation and double pulse stimulation, in the hippocampal CA1 The radiation layer induces and records basal fEPSPs, LTP induced by tetanic stimulation, and pairedpulse facilitation (PPF) induced by paired pulses.
Results: (1) A beta 25-35 did not affect the basic synaptic transmission, but it had a significant inhibition effect on LTP induced by high frequency stimulation. After HFS, 0 min, 30 min, 60 min were given, and the fEPSPs amplitude in the control group was increased to 176.8 + 4.1%, 150.6 + 2.8% and 141.3 +, and the fEPSPs amplitude of 4 nmol A beta 25-35 groups was only 0. The results showed that the A beta 25-35 fragment could obviously suppress the LTP (P0.01) (P0.01) (2) of the hippocampal region (P0.01) (2) to be given to 0.2 nmol Colivelin alone. The basic synaptic transmission and LTP had not changed obviously. It was 178.5 + 2.9%, 139.9 + 2.9%, 138.5 + 2.1%, respectively, after giving HFS, after giving HFS, 139.9 + 2.9%, 138.5 + 2.1%. The value of A beta 25-35, in the 0.2 pmol Colivelin+A beta 25-35 groups, is divided into 164.5 + 1.8%, 122.4 + 1.9%, 110.3 + 1.1%, and in the 2 pmolColivelin+ A beta group 25-35, the values are respectively 166 + 2.3%, 132.2 + 122.4, respectively. The values of min, 30 min and 60 min were higher than that of A beta 25-35 LTP (P0.01) alone, and the difference was dose-dependent. It showed that Colivelin could reverse the dose dependence of A beta against LTP. (3) there was no significant difference between the control group and the PPF of the experimental groups, suggesting that the effects of all kinds of drugs on LTP were not through the presynaptic machine. The system is realized.
Conclusion: intramuscular injection of A beta 25-35 can inhibit the LTP in the hippocampus of rats. The use of Colivelin alone will not affect the basic transfer function of the hippocampus and LTP, but the preconditioning before A beta 25-35 can partly improve the inhibitory effect of A beta 25-35 on LTP, and this effect is dependent on the dose of dose.
In conclusion, this study uses two experimental methods, Morris water maze and LTP, to prove that Colivelin can effectively antagonize the behavior of A beta and the injury of LTP in the hippocampus. Therefore, this study provides a new clue and idea for the prevention and treatment of AD.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.16

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