【作者】 张丽欣；

【导师】 张会珍；

【作者基本信息】 河北医科大学 ， 针灸推拿学， 2008， 硕士

【摘要】 目的:血管性痴呆(vascular dementia,VD)是指各种脑血管疾病引起的脑功能障碍而产生的获得性智能损害综合征。属中医学老年呆病范畴。近年来,VD的患病率越来越高,已成为严重影响人们生活质量的重要因素。因此,深入探讨VD的病理机制,开发和研制防治VD的有效方法具有非常重要的意义。本研究根据中医对本病的传统认识和现代研究成果,认为气虚血瘀、瘀阻脑络为本病的基本病机,以“益气活血、祛瘀通络”为基本法则,选用百会、大椎、膈俞、足三里组方,采用双侧颈总动脉结扎的方法复制VD小鼠模型,观察电针对拟血管性痴呆小鼠行为学、海马组织病理形态学、脑组织氧自由基代谢及脑组织海马细胞凋亡等指标的影响,以探讨其作用机制,从而为进一步研发临床防治VD的有效治法提供依据。方法:第一部分电针对拟血管性痴呆小鼠行为学及脑组织海马病理形态学的影响参照文献采用双侧颈总动脉结扎的方法复制缺血再灌注血管性痴呆小鼠模型。选用健康雄性昆明小鼠80只,将动物随机分为假手术组、模型组、电针组、尼莫地平对照组。造模时将小鼠用10%水合氯醛(0.35g/kg)腹腔注射麻醉,颈正中部常规消毒后切口,分离双侧颈总动脉,套“4”号丝线扣,拉紧丝扣,阻断血流20min,同时在距尾尖1cm处剪断放血约0.3ml,热凝止血。松开丝扣使血液灌注10min后,再次阻断血流20min,第2次再灌后观察30min,缝合皮肤。其中假手术组只分离颈总动脉,穿线但不结扎,尾部不放血,观察时间与其它组相同。术后当天动物苏醒后开始治疗。电针组选百会、大椎、足三里、膈俞穴,用30号0.5寸毫针刺入,接电针仪,采用疏密波,频率2-80Hz,强度以动物肢体轻轻颤动,但不挣扎嘶叫为度,每次10min;尼莫地平对照组灌服尼莫地平液(30mg/kg体重);除电针组外,其余各组均采用同一体位固定10min;除尼莫地平组外,其余各组均灌服生理盐水(10ml/kg体重)。治疗由同一操作者操作,每日1次,连续治疗15天。各组均于治疗结束后,进行行为学检测(采用跳台法),观察小鼠学习记忆能力。然后断头处死小鼠,冰盘上快速剖取全脑,弃去嗅球及小脑。将左侧脑组织置于10%甲醛溶液中固定,常规HE、Nissl染色,光镜下观察脑组织海马CA1区的形态变化。第二部分电针对拟血管性痴呆小鼠脑组织氧自由基代谢的影响造模方法、分组及治疗过程同第一部分。断头处死小鼠,取右侧脑组织制成10%匀浆,4℃3600r/ min离心5min,提取上清液,用双缩脲法测定蛋白含量,采用硫代巴比妥酸比色分析法测定脑组织MDA含量,黄嘌呤氧化酶法测定脑组织SOD活性。第三部分电针对拟血管性痴呆小鼠脑组织海马细胞凋亡的影响脑组织海马细胞凋亡检测采用末端脱氧核糖核酸介导生物素化脱氧尿嘧啶缺口末端标记(Tunel)法。造模方法、分组及治疗过程同第一部分。断头处死小鼠,将左侧脑组织置于10%甲醛溶液中固定,常规石腊包埋、切片、脱腊,蛋白酶K消化;加样、反应、显色等步骤严格按试剂盒说明书进行;反应结束后,常规封片、镜检并做图像分析,观察各组凋亡细胞数。结果:第一部分电针对拟血管性痴呆小鼠行为学及脑组织海马病理形态学的影响1电针对VD模型小鼠行为学的影响模型组小鼠跳台试验的反应时间(51.87±7.79)延长,潜伏期(72.99±10.53)缩短,错误次数(8.00±1.96,8.19±2.21)明显增加,与假手术组(16.72±3.26,262.10±35.29,1.52±0.23,1.38±0.20)比较,具有统计学意义(P<0.01)。电针组(43.10±4.38, 167.71±23.36,4.54±0.54,4.08±0.52)、尼莫地平组(47.31±7.01, 130.17±27.79,5.14±0.78,5.11±0.74)与模型组比较,反应时间缩短,潜伏期延长,错误次数明显减少,有显著性差异(P<0.05或P<0.01)。电针组与尼莫地平组相比,学习成绩之间无显著性差异(P>0.05),记忆成绩相比差异有统计学意义(P<0.05或P<0.01)。2电针对VD模型小鼠海马组织病理形态学的影响光镜下形态学观察:假手术组HE染色海马CA1区锥体细胞层结构紧密,层次丰富,细胞核圆而大,核仁清晰;Nissl染色示胞浆内尼氏体丰富,神经纤维密集,排列整齐。模型组HE染色海马CA1区锥体细胞层次不清,细胞明显脱失,细胞结构不完整,排列稀疏、紊乱,细胞核体积变小、深染,呈核固缩表现;Nissl染色示胞浆内尼氏体消失,神经纤维排列紊乱。电针组及尼莫地平组HE染色海马CA1区细胞结构比较清晰,细胞排列紧密,层次丰富,数量明显多于模型组;Nissl染色显示胞浆内尼氏体比较丰富,仅见少数细胞变性、坏死。其中针刺组略优于尼莫地平组。第二部分电针对拟血管性痴呆小鼠脑组织氧自由基代谢的影响模型组小鼠脑组织MDA含量(10.37±2.19)明显高于假手术组(3.22±0.39) (P<0.01);电针组(3.87±0.57)及尼莫地平组(5.03±0.75)均低于模型组(P<0.01);电针组与尼莫地平组比较,电针组低于尼莫地平组,有统计学差异(P<0.05)。模型组小鼠脑组织SOD活性(13.43±2.44)明显低于假手术组(35.15±4.11) (P<0.01);电针组(33.35±4.47)及尼莫地平组(30.14±3.06)均高于模型组(P<0.01);电针组与尼莫地平组比较,电针组高于尼莫地平组,有统计学差异(P<0.05)。第三部分电针对拟血管性痴呆小鼠脑组织海马细胞凋亡的影响模型组(32.77±6.58)可见大量凋亡细胞,假手术组(8.74±1.58)仅见少量散在凋亡细胞,模型组明显多于假手术组(P<0.01);电针组(25.79±5.57)及尼莫地平(28.75±5.70)组均可见少量散在凋亡细胞,与模型组比较,有统计学意义(P<0.05或P<0.01),电针组与尼莫地平组相比,无统计学意义(P>0.05)。结论:电针对VD模型小鼠具有治疗作用,可显著改善模型小鼠学习记忆能力;减轻因缺血再灌注所造成的海马病理损害;可降低VD模型小鼠脑组织MDA的含量,提高SOD的活性,调节和改善氧自由基代谢,具有增强抗氧化能力的作用;抑制VD模型小鼠脑组织海马CA1区细胞的过度凋亡,促进受损神经元的修复,减轻缺血再灌注损伤。更多还原

【Abstract】 Objective: Vascular Dementia (VD hereinafter) is a kind of acquired intelligence damage syndrome caused by brain dysfunction resulted from various kind of Vascular diseases. It subordinates to the disease scope of dementia of old age in the light of TCM (tradditional Chinese medincine). In the recent years, the prevalence of vascular dementia is on the rise and affects seriously the people’s working ability as well as living quality. We made deep research on the etiology and pathogenesis of VD according to the traditional knowledge of Chinese medicine and modern research achievement and concluded that the fundamental pathogenesis of VD lies in Qi deficiency, blood stasis and collateral-obstruction of the brain. Therefore, we made the treatment principle of“strengthening qi, promoting blood flow”, clinical treatment with electroacupuncture, dazhui、baihui、geshu and zusanli are selected as the main points, we got highly effect in treating VD. In order to make probe into its treatment action mechanism, the experiment observed its effect on praxiology, pathomorphology, oxygen free radicals, cell apoptosis of hippocampus. Methods:Part One The effect of electroacupuncture on praxiology and cerebral pathomorphology of VD miceWe duplicated the model of VD mice with the cerebral ischemic-reperfusion operation with the ligation both side carotid artery. 80 Kunming mice were divided into four groups at random, which were sham-operation group, model group, electroacupuncture group and Nimodipine group. After anesthesia by abdominal injection(350mg/kg)with chloral hydrate, the mouse was fixed on the operation platform in supine, after routine sterilization, the incision was done at the center of neck and bilateral common carotid arteries were separated. And the blood flow was occluded with No.4 silk suture for 20min, meanwhile, 1cm length of tail was clipped off to bleed about 0.3mL and the bleeding was stopped with heat cure. Then released for 10min and occluded again for another 20min, after the second reperfusion, the mouse was observed for 30min and the incision was sutured. In sham-operated group, bilateral common carotid arteries were only separated but not occluded and the tail was not clipped off to bleed. The observing time was same to the other groups.The treatment started after the mouse analepsia that day, dazhui、baihui、geshu and zusanli are selected as the main points in the electroacupuncture group, with No.30 acupuncture pin of 0.5cun pricked into, use electric acupuncture apparatus, adopt thin dense wave with the frequency of 2~80Hz, at the strength of the mouse limbs trill lightly but not struggle and vocalization, 10min every time; The Nimodipine group were given Nimodipine(30mg/kg); Except the electroacupuncture group, the other groups were fixed with same position 10min; Except the Nimodipine group, the other groups were given physiological saline(10ml/kg). The treatment run by same person, one time per day, lasted 15 days. To observe the learning and memory ability of the mouse, the praxiology experiment (jumping stand)was performed on the end of the treatment. After that, the mice were beheaded and the brains were taken out on the ice plate, the olfactory bulb and cerebellum were thrown away. The left part of which was fixed by 10% formaldehyde solution. The tissue was stained by HE(Hematoxylin)and Nissl. We observed constitutional morphologic changes of cerebral tissue under light microscope.Part Two The effect of electroacupuncture on oxygen free radicals in brain of VD miceThe way to make model and the treatment were same to part one. The mice were beheaded, the right brain was make into 10% homogenate. The homogenate was centrifugated for 5min at 4℃, 3600r/min, the supernatant was extracted. The experiment used biuret method to determin protein level, and thio-barbituric acid compared color analytical method to detect the MDA content, xanthine oxidase method to detect the activity changes of SOD in cerebral tissue of Kunming mice.Part Three The effect of electroacupuncture on Apoptosis of hippocampus cell in brain of VD micewe used the method of Terminal Deoxynucleotidyl Transferase-mediated dUTP-Nick End-labeling (TUNEL)to label the apoptosis cells. The way to make model and the treatment were same to part one. The mice were beheaded, the left part of which was fixed by 10% formaldehyde solution. Embed with paraffin、slice、take off paraffin at rule, digest Proteinase K; Application of sample、reaction、coloration were conducted strictly according to the directions in its kit; Mounting、test under microscope and image analysis at rule after reaction, observe the number of apoptosis cell in each group.Results:Part One The effect of electroacupuncture on praxiology and cerebral pathomorphology of VD mice1 Results of praxiology experimentModel group had longer reaction time(the time of jumping up the stand)(51.87±7.79), shorter latency time(the time of jumping down the stand)(72.99±10.53)and more wrong times(8.00±1.96,8.19±2.21)than sham-operation group (16.72±3.26,262.10±35.29,1.52±0.23,1.38±0.20)(P<0.01). At the same time, electroacupuncture group(43.10±4.38,167.71±23.36,4.54±0.54,4.08±0.52)and Nimodipine group(47.31±7.01,130.17±27.79,5.14±0.78,5.11±0.74)had shorter reaction time, longer latency time and less wrong times than model group(P<0.05 or P<0.01). Compared with Nimodipine group, the electroacupuncture group had better memory(P<0.05 or P<0.01), but had no difference in learning(P>0.05).2 The effect of electroacupuncture on Hippocampus pathomorphology in brain of VD miceGeneral morphology of brain tissues was shown under light microscope: In the sham-operated group, the hippocampal pyramid cells in CA1 area were closely arranged by HE staining, the karyons were round and big and the nucleoli were clear; Rich Nissl bodies were seen by Nissl staining and dense, well-arranged nerve fibers were observed as well. In the model group, the layers of hippocampal pyramid cells were not obvious by HE staining, the cells were evidently lost and the cell line became indistinct, the structure of many cells turned incomplete and the gap emerged among confused, sparsely arranged cells; nucleus volume shrank,dark staining, unclear structure, karyopyknosis appeared; Nissl bodies inside cytoplasm disappeared and nerve fibers were in disorder by Nissl staining. In all treatment groups, the cell lines in CA1 area of hippocampus were relatively clear by HE staining, the arrangement of cells was relatively compact and the amount of cells increased obviously; Nissl bodies inside cytoplasm were relatively abundant seen by Nissl staining and only a few cells were found degenerative and necrotic. The electroacupuncture group was similar and slightly better than that in the Nimodipine group in both HE and Nissl staining.Part Two The effect of electroacupuncture on oxygen free radicals in brain of VD miceThe result manifested that the content of MDA in cerebral tissue of model group(10.37±2.19)was raised significantly than that in sham-operation group(3.22±0.39)(P<0.01); At the same tine, the MDA content of electroacupuncture group(3.87±0.57)and Nimodipine group(5.03±0.75)were lower than that of model group(P<0.01); The MDA content in cerebral tissue of electroacupuncture group was lower than that Nimodipine group(P<0.05).The result manifested that the activity of SOD in cerebral tissue of model group(13.43±2.44)was reduced significantly than that in sham-operation group(35.15±4.11)(P<0.01); The SOD activity of electroacupuncture group(33.35±4.47)and Nimodipine group(30.14±3.06)were higher than that of model group(P<0.01); The SOD activity of cerebral tissue of electroacupuncture group was higher than that of Nimodipine group(P<0.05).Part Three The effect of electroacupuncture on Apoptosis of hippocampus cell in brain of VD miceThe sham-operation group(8.74±1.58)had a small quantity of apoptosis cell in disorder, the model group(32.77±6.58)had a great quantity of apoptosis cell, it was higher significantly than that in sham-operation group(P<0.01); The electroacupuncture group(25.79±5.57)and Nimodipine group(28.75±5.70)also had a small quantity of apoptosis cell, they were lower than that of model group(P<0.05 or P<0.01), but had no difference between themselves(P>0.05).Conclusion : The experiment demonstrated that electroacupuncture had therapeutical effect on VD mice and relieved pathological lesion of cerebral ischemia-reperfusion, and it could improve learning and memory ability of VD mice significantly; Electroacupuncture could reduce MDA content and increase the activity of SOD in cerebral tissue of VD mice significantly, it could resist the oxidative injury to alleviate the physical injury from lipid peroxidation and free radical; Electroacupuncture could restrain excessive apoptosis of hippocampus cell in brain of VD mice, At the same time it promoted nerve cell.更多还原