【作者】 沈瑞乐；

【导师】 滕军放；

【作者基本信息】 郑州大学 ， 神经病学， 2007， 硕士

【摘要】 目的与背景慢性脑缺血是血管性痴呆(vascular dementia，VD)和阿尔茨海默病(Alzheimer’s disease，AD)发病的重要基础。近来研究证明，AD和VD均存在明显的血管因素，而脑血流低灌注在痴呆的发病进程中的作用日益引起重视，因此研究其发病机制对慢性脑缺血相关性疾病的临床防治具有普遍意义。慢性脑缺血所致的神经损伤主要表现为学习和记忆等认知功能损害。海马缺血性损伤是其主要的病理基础。慢性脑缺血导致海马功能改变包含两个主要方面的作用：神经细胞的损伤和延迟性胆碱能功能的缺损。中枢胆碱能神经有促进学习记忆、激活脑电、维持觉醒、参与精神意志活动等重要生理功能。胆碱乙酰转移酶(choline acetyltransferase，Chat)常作为研究胆碱能神经元的特殊标志。丁基苯酞(dl-n-butylphthalide,NBP)是治疗脑缺血的一种新药。有研究表明NBP可改善小鼠全脑缺血脑能量代谢，增加局部脑缺血大鼠缺血区的脑血流，改善大脑中动脉结扎(middle cerebral artery occlusion，MCAO)大鼠学习记忆障碍。本文通过免疫组化、反转录聚合酶链式反应(Reverse Transcriptase Polymerse Chain Rection，RT-PCR)、斑点免疫结合实验三种方法观察大鼠海马区Chat的变化，旨在探讨NBP对慢性缺血引起的认知功能障碍的影响及其作用机制。材料方法1．实验动物与分组健康Wistar大鼠150只，随机分为5组，每组30只，A组：对照组(假手术+溶剂)；B组：单纯缺血组(手术+溶剂)；C组：缺血低剂量治疗组(手术+低剂量NBP+溶剂)；D组：缺血高剂量治疗组(手术+高剂量NBP+溶剂)；E组：预防组(中等剂量NBP+溶剂+手术)。2．模型的制作钝性分离单纯缺血组和缺血高、低剂量治疗组大鼠双侧颈总动脉，用0号手术线分别结扎其近心端及远心端；分离对照组双侧颈总动脉但不结扎。3．标本的制作手术3月后，所有大鼠经心脏灌注后，断头取脑，部分用4％的多聚甲醛固定后做石蜡切片，供做免疫组化用；部分放入超低温冰箱，以备RT-PCR及免疫斑点表达。4．实验室检测方法应用免疫组化、反转录聚合酶链式反应、斑点免疫结合实验等技术。5．统计学方法应用SPSS 13.0统计软件(SPSS Inc)进行统计学分析。符合正态分布且方差齐，不同组之间的计量资料的比较应用单因素方差分析，否则用非参数检验。检验水准α=0.05。结果1．实验动物一般情况。术后大鼠均出现精神萎靡，反应迟钝，进食减少。术后1天，A组精神恢复；B、C、D、E组改善不明显。3-5天B、C、D、E组逐渐恢复，1周后反应、进食正常。2．Chat免疫组化染色各组海马区Chat阳性神经元数目进行观察并统计：B组与A组相比，Chat的表达明显降低，差异有统计学意义(P＜0.05)；C组、D组、E组与B组相比Chat的表达明显增加，差异有统计学意义(P＜0.05)；D组与C组、E组相比Chat表达增加，且差异具有统计学意义(P＜0.05)。E组与C组差异不具有统计学意义(P＞0.05)。3．ChatmRNART-PCR的结果各组海马区ChatmRNA光密度值与ActinmRNA光密度值的比值进行观察并统计：B组与A组相比，Chat的表达明显降低，差异具有统计学意义(P＜0.05)；C组、D组、E组与B组相比Chat的表达明显增加，差异有统计学意义(P＜0.05)；D组与C组、E组相比Chat表达增加，且差异具有统计学意义(P＜0.05)；E组与C组差异无统计学意义(P＞0.05)。4．Chat斑点免疫结合实验的结果各组海马区Chat免疫斑点的灰度值进行观察并统计：B组与A组相比，Chat的表达明显降低，差异有统计学意义(P＜0.05)；C组、D组、E组与B组相比Chat的表达明显增加，差异有统计学意义(P＜0.05)；D组与C组、E组相比Chat表达增加，且差异具有统计学意义(P＜0.05)。E组与C组差异无统计学意义(P＞0.05)。结论1．永久性结扎大鼠双侧颈总动脉建立慢性脑缺血模型，接近人类慢性脑缺血的发生过程，手术操作简单，重复性好。2．慢性脑缺血3月后，大鼠海马区胆碱能神经功能明显受损。3．NBP可抑制慢性脑缺血引起的海马区Chat阳性神经元数目减少，提高ChatmRNA的转录水平，增加Chat蛋白的表达，并且NBP还可预防慢性脑缺血后Chat的下降。更多还原

【Abstract】 Background and AimThe chronic cerebral hypoperfusion is the important foundation of vascular dementia (VD)and Alzheimer’s disease(AD).In the recent years,many research has proved that egardless of vascular dementia (VD) or Alzheimer’s disease(AD) has the obvious blood vessel factor. The role of brain hypoperfusion brings to the attention day by day in the process of demented advancement , and proposed the vascular cognitive impairment (VCI) this new concept. Therefore, it is important to study pathogenesis for preventing and treating chronic cerebral hypoperfusion.The chronic cerebral hypoperfusion can cause nerve injury and its main performance is cognitive disorder such as study, memory, and so on. The chronic cerebral hypoperfusion cause the hippocampal change to contain two principal aspects : Nerve cell’s damage and the retardant function impairment of cholinergic nerve. Some researchs report that the impairment of frontal lobe and the cholinergic neurons possibly be the morphological foundation which can cause cognitive disorder,and the cognitive impaired mechanism involves changes of the cholinergic acceptor, and so on . Chat is a rate-limiting enzyme,which control the synthesis of acetycholine( Ach).So it is always regarded as the special signal of cholinergic neurons . D1-3-n-Butylphthalide (NBP) was shown to improve brain energy metabolism in mice with complete cerebral ischemic, to enhance the rCBF of rats with regional ischemia,and to improve impairment of learning and memory of rat with MCAO, and so on. Present study aimed to determine the change of Chat in hippocampus in rats brain by the methods of reverse transcription PCR and Immunohistochemical staining and dot immunobinding assay. By occlusiving bilateral carotid artery, 2VO model of chronic cerebral ischemia was established. The expression of Chat in hippocampus was observed through three methods.The aim of this study is to explore that mechanism of the protective effect of NBP on learning and memory after chronic cerebra ischemia.Materials and methods1. Subjects were divided into 5 groups groups randomly, 20 rats each group. Group A: control group (sham operation plus); group B: ischemia group; group C: ischemia and low dose treatment group; group D: ischemia and high dose treatment group; group E: ischemia and middle dose treatment. In front of operation ,the rats of groupE give NBP according to 80mg/kg fill the stomach every day, and after the operation , continues to fill the stomach for two months according to this dosage.The rats of groups C and D began to receive daily oral dose of 60mg/kg and 120mg/kg NBP (dissolved into 2ml oil) from the 61st day after the operation, which will last a month. The rats of groups A and B received the same volume of oil, which will last the same time.2. Chronic cerebral ischemic rat model was established by permanent occlusion and snip of bilateral common carotid arteries. Rats undergoing permanent bilateral occlusion and snip of the common carotid arteries were distributed into ischemia group or ischemia treatment group. Rats with bilateral common carotid arteries only isolated but not ligated and snipped were into control group.3. After three months , all rats after the heart irrigation, behead take the left brains, fixed makes the paraffin section after 4% paraformaldehyde, for immunohistochemistry staining; The right brains were put in the supercold refrigerator, for RT-PCR and Dot Immunobinding Assay.4. Used the immunohistochemical staining to observe the distribution of Chat immunopositive cells in hippocampus in rats brain ; Used the RT-PCR method to examine ChatmRNA transcription level in hippocampus in rats brain; Used the dot immunobinding assay method to study the expression of Chat protein in hippocampus in rats brain. 5. The data was handled with SPSS13.0(Inc). The diference of each group was compared with one-way analysis of variance,and only values < 0.05 were considered statistically significant.Results1. The conditions of animal All the rats were depressed, reacted slowly, ate less after operation. The next day, sham groups recovered significantly, however the operation groups recovered slowly until 3-5 days. A week later, they were normal.2. The results of immunohistochemical staining for CHAT The number of Chat immunopositive cells in hippocampus was significant decreased in group B comparing with group A. Significant differences were observed between group B and group A. The number of Chat immunopositive cells was markly increased in group C , group D and group E comparing with B group. Significant differences were observed between group C, group D and group E. The number of Chat immunopositive cells was markly increased in group D comparing with group C and group E. Significant differences were observed between group C, group D and group E.No significant differences were observed between group C and group E.3. The results of RT-PCR for CHAT Significant differences were observed between group B and group A.The optical density value was significant increased in group C , group D and group E comparing with B group. Significant differences were also observed between group C, group D and group B. The number was significant increased in group D comparing with group C and group E. And significant differences were observed between group C, group D and group E. No significant differences were observed between group C and group E.4. The results of Chat Dot Immunobinding Assay The grey number was significant decreased in group B comparing with group A. Significant differences were observed between group B and group A.The grey number was markly increased in group C , group D and group E comparing with B group. Significant differences were observed between group C, group D and group B. The grey number was significant increased in group D comparing with group C and group E. Significant differences were observed between group C, group D and group E .However, No significant differences were observed between group C and group E.Conclusions1. Chronic cerebral ischemia rat model was established by permanent occlusion and snip of bilateral common carotid arteries.The progress of the model and chronic cerebral ischemia was almost same. Its operation is simple and repeat well.2. Three months after, significantly functional impairment appeared in hippocampus in rats brain.3. NBP can suppress the diminution of immunopositive cells in hippocampus in rats brain which chronic cerebral hypoperfusion caused, enhance ChatmRNA the transcription level, increase the Chat protein the expression.更多还原