【作者】 苗庆峰；

【导师】 张永健；

【作者基本信息】 河北医科大学 ， 药理学， 2004， 硕士

【摘要】 随着人口老龄化的趋势日益明显，老年性痴呆已成为医学上十分关注的问题。老年性痴呆的患病率随年龄的增长而呈指数上升。阿尔茨海默病（Alzheimer’s disease, AD）是老年性痴呆中最常见的类型，其发病较早，一般在60岁之前，又可称为早老性痴呆。老年性痴呆不仅严重影响患者的生活质量，还给家庭和社会带来巨大的经济负担，且目前临床上尚无有效的治疗药物，因此开发治疗老年性痴呆的药物是急待攻破的世界性难题。双苯氟嗪（Dipfluzine，Dip）是由河北医科大学首次合成的一种新型钙拮抗剂，其脂溶性特点决定了Dip可以透过血脑屏障到达脑部发挥作用。实验证明Dip可以阻断细胞外钙离子内流，保证细胞内钙稳定，改善脑微循环，保证细胞的能量和营养供应，对亚硝酸钠和戊巴比妥钠造成的小鼠学习记忆障碍有改善作用，能够通过改善皮质功能紊乱和脑内兴奋性与抑制性氨基酸释放失调对脑缺血大鼠起脑保护作用。鉴于Dip可以改善脑功能，设想其可能对老年性痴呆有一定的作用。本实验采用腹腔注射三氯化铝（AlCl3）制备大鼠AD模型以及选用自然衰老大鼠，观察Dip对两种大鼠的脑保护作用，并进行机制方面的研究。实验内容如下： <WP=5>一、Dip对AD大鼠的脑保护作用及其机制目的：采用跳台试验观察Dip对AD模型大鼠学习记忆能力的影响，并通过测定血清及脑组织中脂质过氧化产物含量或活性以及神经细胞胆碱乙酰转移酶（ChAT）表达，探讨Dip对AD大鼠脑保护作用机制。方法：选用350～400g健康雄性SD大鼠，随机分为5组：(1)正常对照组；(2)模型对照组；（3）尼莫地平阳性对照组（Nimodipine, Nim 5.0mg/kg）；（4）Dip 10.0mg/kg组；（5）Dip 3.0mg/kg组。除正常对照组给予等比例的生理盐水外，其余各组每天腹腔注射1.25% AlCl3生理盐水溶液4ml/kg体重，连续60天。实验开始后前3天，各组分别以半量给予，每连续注射3天，间隔1天。末次给药后1小时进行学习训练，记录大鼠受到电刺激后跳上平台的时间（反应时间）、第一次跳下平台的时间（跳台潜伏期）、5分钟内大鼠遭受的电击次数（错误次数）以及5分钟内受电击的总时间（累积电击时间），作为学习成绩；24小时后重复测试，作为记忆测试成绩。眼球后静脉丛取血并制备脑组织匀浆，按试剂盒操作说明分别测定血清和脑组织中总抗氧化能力（T-AOC）、超氧化物歧化酶（SOD）活性及丙二醛（MDA）含量。大鼠断头取脑，4%多聚甲醛固定，常规梯度酒精脱水，二甲苯透明，石蜡包埋，切片，HE染色，观察海马神经元损伤状况。免疫组化观察各组海马神经元ChAT表达情况。结果：1、跳台试验结果表明，模型对照组大鼠学习记忆能力较正常对照组明显下降，其反应时间及累积电击时间均长于正常对照组（P<0.05或P<0.01），跳台潜伏期明显缩短（P<0.01），错误次数增多（P<0.05或P<0.01）；Dip <WP=6>10.0mg/kg及Nim可明显缩短模型大鼠学习记忆测试中反应时间及累积电击时间，延长跳台潜伏期，减少错误次数（P<0.05或P<0.01）；Dip 3.0mg/kg可使模型大鼠学习记忆测试的反应时间及累积电击时间缩短、跳台潜伏期延长（P<0.05或P<0.01），学习测试中错误次数明显少于模型对照组（P<0.01），并且记忆测试中错误次数较模型对照组有减少的趋势，但无统计学意义（P>0.05）。2、测定血清及脑组织匀浆生化指标表明，模型对照组大鼠血清及脑组织中T-AOC和SOD活性较正常对照组有所下降（P<0.05），而MDA含量明显升高（P<0.01）；Dip10.0mg/kg组、Nim组与模型对照组比，T-AOC和SOD活性均升高（P<0.05或P<0.01），而MDA含量降低（P<0.05或P<0.01）；Dip 3.0mg/kg组与模型对照组比，血清中T-AOC升高（P<0.05）而MDA明显降低（P<0.01），脑组织中MDA含量降低（P<0.05），但血清和脑组织中SOD活性及脑组织中T-AOC与模型对照组无明显差别（P>0.05）。3、病理结果显示，正常对照组大鼠海马结构正常，海马锥体细胞排列整齐紧密，核仁清晰可见，CA1区的正常锥体细胞数为180±10个/mm；模型对照组大鼠海马结构被破坏，部分神经元细胞核浓染，固缩变形，正常神经元数目减少（118±12个/mm）；Nim及Dip10.0mg/kg组 CA1区正常锥体细胞数分别为154±14个/mm和159±14个/mm，坏死细胞明显减少，海马损伤得到改善；Dip 3.0mg/kg组亦可见轻度的改善作用。4、ChAT免疫组化可见，正常对照组海马及皮质组织中有大量的阳性神经元，胞浆被染成黄褐色，表达量<WP=7>较高；模型对照组免疫阳性神经元明显减少，且表达量少；而Nim和Dip10.0mg/kg均可以增加免疫阳性细胞数及表达量；Dip 3.0mg/kg对ChAT表达无明显的影响。结论：Dip可以提高铝致AD大鼠的学习记忆能力，保护海马神经元免受铝损伤，这种作用可能与其提高机体抗氧化能力及增强胆碱能神经功能有关。二、Dip对自然衰老大鼠的脑保护作用及其机制目的：观察Dip对自然衰老大鼠学习记忆能力、脑细胞形态的影响，并通过测定血清中脂质过氧化产物含量或活性、神经细胞内游离钙浓度及神经细胞凋亡百分率，研究Dip对自然衰老大鼠的脑保护作用机制。方法：采用21月龄雄性大鼠，随机分为3组：（1）模型对照组；（2）Nim 5.0mg/kg组；（3）Dip 5.0mg/kg组；另更多还原

【Abstract】 With the tendency of aging, senile dementia has become the focus in the medical field. The morbidity of senile dementia is increased remarkably with the growing age. Alzheimer’s disease (AD）, the most common of the senile dementia, has serious effect on both the work and life of people. But there is no effective drug for treating senile dementia at present. Dipfluzine (Dip), a novel calcium antagonist, was first synthesized by Hebei Medical University. It is lipophilic, which enables it to cross the blood-brain-barrier and achieve effective drug concentration in the cerebrospinal fluid. Dip could inhibit calcium influx and maintain intracellular calcium homeostasis. It could improve cerebral microcirculation and assure supply of energy and nutrition in cells. It could improve the amnesia induced by sodium nitrite and sodium pentobarbital in mice and protect the ischemic brain in rat by reducing the disturbance of cortical function and the imbalance between excitatory and inhibitory amino acids.The present study is undertaken to observe if Dip possesses the protective effects on the brain in naturally aged rats and AD rats induced by intraperitoneal injection with AlCl3 and explore its mechanisms. The concrete experiments are as <WP=11>follows:1. Protective effects of dipfluzine on the brain in AD ratsObjective: To study the protective effects of Dip on the brain in AD rats by measuring learning and memory ability, some biochemistry indexes in brain tissue and blood serum and expression of choline acetyltransferase (ChAT) in the brain.Methods: Healthy Male SD rats (350-400g) were randomly divided into 5 groups: (1) Control group; (2) Model group; (3) Nim group (5.0mg/kg, ig); (4) High dose Dip group (10.0mg/kg, ig); (5) Low dose Dip group (3.0mg/kg, ig). Rats in Control group were treated by intraperitoneal injection with saline 4ml/kg while the rats in the others were treated by intraperitoneal injection with 1.25%AlCl3 by the same volume for 60 days. The step-down avoidance test was used to observe the learning and memory ability of rats. Biochemisty method was used to determine SOD, T-AOC and MDA in brain tissue and blood serum. All the rats were killed by decapitation and the brains were removed and immersed in 4% pareformaldehyde, then the brains were processed and embedded in parafin. Coronal sections were taken from the dorsal hippocampal and stained with HE. The sections were examined with the light microscope and neuronal damage was observed. The expression of ChAT in neuron was observed by immunohistochemisty.Results: 1. The learning and memory ability of the rats in <WP=12>model group was significantly lower than that in control group. The reactive time and the time of stimulation of the rats in model group were significantly longer than that in control group, and the number of errors was more and the step down latency was significantly shorter as well. Both Nim and Dip 10.0mg/kg improved the spatial disorder of the model rats remarkably, while Dip 3.0mg/kg could improve some index only. 2. Compared with control group, the content of MDA in the brain tissue and blood serum was increased obviously and the activities of SOD and T-AOC were decreased clearly in model group. In the groups treated with Nim and Dip, the content of MDA was decreased while activities of SOD and T-AOC were increased in different degree. 3. Observation by light microscope: The hippocampal structure of rats in control group was normal as well as the morphology of pyramidal neurons and the number of the normal hippocampal cells in CA1 area was 180±10. In model group, the hippocampal structure was destroyed, and almost all pyramidal neurons were either shrinked or dissolved and the number of the normal hippocampal cells in CA1 area was lower(118±12) than that in control group. Nim and Dip 10.0mg/kg lightened the injury in different degree and decreased the number of dead neurons and the number of the normal hippocampal cells in CA1 area was 154±14 and 159±14. Dip 3.0mg/kg could improve the damage of brain更多还原