【作者】 李彩霞；

【导师】 李建章； 刘其强；

【作者基本信息】 郑州大学 ， 神经病学， 2004， 硕士

【摘要】 慢性脑缺血是指各种原因导致的长期的脑灌流不足，促使脑发生组织病理及生化改变，是血管性痴呆、Binswanger病、Alzheimer病等多种疾病发展过程中的一个共同病理过程。随着我国老龄人口比例增大，预防和治疗主要由慢性脑缺血带来的老年性疾病就具有了重大社会意义。脑组织慢性缺血时，神经突触发生了病理性改变，特别是胆碱类突触改变明显；自由基增多，其清除剂超氧化物岐化酶(SOD)下降，脂质过氧化物增多，其毒性代谢产物丙二醛(MDA)也相应升高，而MDA是一种毒性物质，可致细胞膜过氧化损伤，脑组织神经胶质细胞增多，其表达的胶质原纤维酸性蛋白(GFAP)即其标志性蛋白明显增多。达纳康是一种标准化银杏叶萃取物，其主要成分是黄酮糖苷、银杏苦内酯和白果内酯。药理学研究表明，达纳康能改善脑循环，增加脑的血流量、改善缺血脑的能量代谢、清除自由基、保护细胞膜的结构和功能的完整性，对缺血状态下的神经细胞有一定的保护作用。本实验以突触的超微结构、SOD、MDA、GFAP为指标观察达纳康对慢性脑缺血大鼠的脑保护作用，旨在为达纳康用于防治老年相关性疾病提供理论依据。 材料与方法：(1)选健康Wistar大鼠80只，雌雄不拘，随机分为4组，每组20只。A组：对照组(假手术+溶剂)；B组：单纯用药组(假手术+达纳康+溶剂)；C组：单纯缺血组(缺血+溶剂)；D组：缺血用药组(缺血+达纳康+溶剂)。非用药的对照组和单纯缺血组予0.5％羧甲基纤维素钠溶液3毫升，用药组予等量0.5％羧甲基 郑州大学2004届硕士研究生毕业论文达纳康对慢性脑缺血大鼠的脑保护作用观察纤维素钠溶解均匀的达纳康(Zoom眺g)溶液。(2)模型的制作:缺血组双重丝线结扎双侧颈总动脉，对照组和单纯用药组仅分离双侧颈总动脉而不结扎。常规条件下饲养7周予以处死取标本。(3)电镜组鼠脱臼法处死后断头取脑，取海马部位标本IXI xZmm3大小放入4%戊二醛溶液固定，再经一系列后期处理，超薄切片(片厚约50nm)，H一7500型电镜80KV下观察;生化指标组:脱臼法处死鼠，断头取脑研磨，制成10%脑组织匀浆，测SOD、MDA吸光光度值;免疫组化组:快速经主动脉生理盐水冲洗后4%多聚甲醛内固定，断头取脑放入4%多聚甲醛固定，常规石蜡包埋、切片，切片厚度约3腼，采用HE染色及免疫组化SABC法，检测海马CAI区GFAP阳性细胞数。(4)应用sPSS 10.0统计软件进行数据处理。统计方法用单因素方差分析并用LSD法进行两两比较，取a=0 .05作为检验水准。 结果:(1)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l天假手术组鼠精神恢复，反应灵敏，进食增多。灌胃期间缺血组改善一直不明显。 (2)电镜下见对照组和单纯用药组突触正常，无肿胀、间隙模糊。单纯缺血组见前、后膜肿胀，突触间隙模糊，突触后膜致密物增厚。缺血用药组突触前、后膜肿胀不明显。 (3)SOD:A组:151.58士6.79:B组:156.90士6.02:C组:123.71士7.11:D组:134.49士8.15:MDA:A组:5.34士0.67:B组:5.14士0.58:C组:12.92士0.87:D组:8.14士0.73。两指标A、B组间均无显著性差异(P>0 .05)，其余任两组间均有显著性差异(P<0 .01)，表明达纳康对正常大鼠脑组织SOD活性和过氧化产物MDA的生成无明显影响;大鼠慢性脑缺血后SOD活性明显下降，MDA生成明显增多;达纳康治疗后SOD活性明显上升，MDA生成明显减少。 (4)海马CAI区GFAP阳性细胞数:A组:30.90士3.67:B组:30.50士4.19:C组:51.30士7.94;D组:38.10士4.89。A、B组间无显著性差异(P>0 .05)，其余任两组间均有显著性差异(P<0.01)，表明达纳康对正常大鼠海马星形胶质细胞无明显影响，慢性脑缺血后星形胶质细胞大量增生、肥大，达纳康治疗后星形胶质细胞增生、肥大明显减轻。 (5)各组病理切片HE染色观察:A、B组见少量神经元变性、死亡，细胞膜核膜溶解，大部分神经元细胞膜、核膜完整，形态正常。C组见大量变性、死亡细胞，细胞膜、核膜溶解、染色质消失。D组见变性死亡细胞较C组明显减少。说明达纳康郑州大学2004届硕士研究生毕业论文达纳康对慢性脑缺血大鼠的脑保护作用观察对正常大鼠神经元无明显作用，慢性脑缺血后大量神经元变性死亡，达纳康治疗减少了神经元变性死亡。 结论:(l)本实验动物模型采用较为常用的双侧颈总动脉永久性结扎，保证鼠脑不完全性缺血，与人类慢性脑缺血相似。该模型简便有效，适合用来研究慢性脑缺血的病理形态学特点及进行疗效观察。 (2)大鼠慢性脑缺血时，海马CAI区突触病态性改变，SOD活性明显下降，MDA大量产生，GFAP阳性星形胶质细胞大量增生、肥大，脑组织损害严重。 (3)达纳康能减轻大鼠慢性脑缺血时海马CAI区突触损害，增强SOD活性，抑制MDA产生，减少星形胶质增生肥大数目。对正常大鼠无明显影响。 (4)达纳康对大鼠慢性脑缺血有明显保护作用，能明显减轻慢性脑缺血带来的损害，为达纳康在临床应用、防治老年相关性疾病提供了理论依据。更多还原

【Abstract】 Chronic cerebral hypoperfusion is chronic cerebrovascular insufficiency for a long time because of all kinds of reasons. It brings out histopathological and biochemical changes in brain ,which is a common pathological way in the period of many disease such as vascular dementia . Binswanger disease . Alzheimer disease .With the ratio of senile increasing in our country, prevention and treatment to diseases mainly from chronic cerebrovascular insufficiency is increasingly important. In the situation of cerebral hypoperfusion, pathological changes take place in nerve synapse, especially in cholinergic synapses. At the same time, free oxidate increases, SOD, its clearer, discreases, LPO increases with its toxic output -MDA increasing relatively. MDA is a kind of toxic matter, which can overoxidate cell membrane. Astrocytes increased, GFAP, their symbol protein, increased too. Tanakan is standard extract of ginkgo biloba. The main ingredient is ginkgolide and bilobalide. The research indicates that tanakan can improve cerebrovascular cycle, improve energy metabolism, clear free oxidate, protect the intact of structure and ability of the cell membrane. Therefore ,it protects the neurons in hypoperfusion .The experiment research the protective effect of tanakan to cerebral hypoperfusion by observing microstructure of synapse . SOD. MDA. GFAP to support prevention and therapy senile diseases with tanakan.Materials and methods: (1) Selected 80 Wistar rats and distributed to 4 groups4randomly, regardless of male or female, 20 rats each group. Group A: controlled (sham operation plus solvent); group B: only treated (sham operation plus tanakan plus solvent); group C: only hypoperfusion (hypoperfusion plus solvent); group D: hypoperfusion and treatment (hypoperfusion plus tanakan plus solvent). Rats underwent permanent bilateral occlusion of the common carotid arteries (2VO) in groups C and D. Bilateral common carotid arteries were only isolated but not ligated in groups A and B. groups A and C swallowed 0.5% carboxymethyl cellulose sodium solution 3ml, groups B and D swallowed the same amount of tanakan (200mg/mg) disolved by 0.5% carboxymethyl cellulose sodium solution. All the rats after were raised in a usual way for 7 weeks, were decapitated. Hippocampus (1 1 2mm)of the rats used to be observed were taken out, then put into solution of 4% glutaral, after a series of treatments, insected to 50nm thick, observed them under 80 KV H-7500 electrolen. The rats used to detect S MDA were decapitated and taken out brains ,then grilled brains and made 10% solution, at last detect SO MDA . The rats used to immunohistochemical observed were shot saline in brain arties then shot with 4% polyformaldehyde solution to be fixed, sacrificed by decapitation, the brain tissue was removed, embed in paraffin and made immunohitochemical sections(3um thick).Using HE staining and immuniohistochemical SABC method, the number of GFAP -positive cells were detected. The data was handled with SPSSIO.O statistic software. The difference of every two groups was compared with one-way analysis of variance and LSD method, significant level is a =0.05.Results: (1). All the rats were in low spirits, reacted slowly, ate little after operation. The next day, sham groups recovered in spirits, reacted quickly and ate more, but hypoperfusion groups improved insignificantly in the period of treatment.(2). Synapes of groups A, B are good observed by electrolen, no swell at membranes of synapses and no blur in gaps between synapses .But in group C, the membranes of synapses are swollen, the gaps between synapes are blurry and matter increased at the promembrane. The membranes of synapses of group D are insignificantly swollen.(3). SOD: group A: 151.58+6.79 ; group B: 156.90+6.02; group C: 123.71 ?.11; group D: 134.49 + 8.15. MDA: group A: 5.35?.67; group B: 5.14?.58; group C: 12.92?.87; group D: 8. 14 ?.73. The difference is significant (P<0.01) between every two groups except groups A and B in SOD and MDA, which indicated that tanakan has n更多还原