【作者】 朱书秀；

【导师】 孙国杰；

【作者基本信息】 湖北中医学院 ， 针灸推拿学， 2009， 博士

【摘要】 目的观察阿尔茨海默病（AD）模型大鼠额叶与海马区的免疫炎性反应、P₃₈MAPK的磷酸化及其超微病理的变化等,以及电针治疗对其的影响,探讨电针防治AD的可能机制,为针刺临床提供新的理论依据。方法110只24月龄SD大鼠,采用Y型水迷路法粗筛,获正常老年大鼠103只,再从103只SD大鼠中随机选出30只,分别归入正常组和假手术组,每组15只。双侧Meynert核注射微量凝聚态Aβ_1-40制备AD动物模型,造模完成后第31天,对大鼠再次进行Y型水迷路法筛选,凡15次测试中正确次数较造模前下降1/3者为造模成功。将造模成功的大鼠随机归入模型组16只,电针组17只。选取“百会”、“太溪”、“足三里”进行电针治疗,每天1次,6天为1疗程,疗程间隔1天,共治疗2疗程。Morris水迷宫行为学实验检测其学习记忆能力,免疫组化SABC法观察大鼠额叶与海马区IL-1β、TNF-α的变化,rt-PCR检测IL-1βmRNA和TNF-αmRNA的表达,Western-blot法观察p-P₃₈MAPK的量,透射电镜观察AD大鼠额叶与海马区神经元超微结构。全部数据输入计算机,用SPSS13.0统计软件包进行处理,实验结果以（（?）±s）表示。结果（1）Morris水迷宫检测发现:在定位航行实验中,第1天,模型组、电针组逃避潜伏期较正常组、假手术组明显延长（P<0.01）;模型组与电针组之间、正常组与假手术组之间无差异（P>0.05）;第2、3、4、5天,各组大鼠均随着训练次数增加,潜伏期越来越短,但潜伏期长短明显不一,正常组和假手术组约在25s内找到平台,模型组约需60s,但电针组在40s内可找到平台,其逃避潜伏期较模型组明显缩短（P<0.01）。在空间探索实验中,模型组与正常组、假手术组相比,原平台象限游泳时间比和原平台象限跨越次数比均显著减小（P<0.01）,10%百分比和30%百分比明显增加（P<0.01）;电针组与模型组比较,原平台象限游泳时间比和原平台象限跨越次数比明显增加（P<0.01）,10%百分比和30%百分比明显减少（P<0.01）。（2）模型组GFAP与CD68阳性细胞、IL-1β与TNF-α表达增加,染色增强,与正常组、假手术组相比有非常显著性意义（P<0.01）;经电针治疗后表达显著减少,与模型组比较,差异有非常显著性（P<0.01）。（3）rt-PCR检测表明:模型组额叶与海马区IL-1βmRNA、TNF-αmRNA的表达较正常组、假手术组增强（P<0.05-0.01）,电针组IL-1βmRNA、TNF-αmRNA表达显著减少,与模型组比较,P<0.05-0.01。（4）Western-blot显示:各组大鼠额叶与海马区均有p-P₃₈MAPK的表达,模型组表达最高,与正常组、假手术组相比具有非常显著性意义（P<0.01）;经电针治疗后额叶与海马区p-P₃₈MAPK表达减少,与模型组相比,差异具有非常显著性意义（P<0.01）。（5）透射电镜下模型组额叶与海马区可见一些神经细胞变性,其内细胞器可见内质网扩张,线粒体肿胀和空泡样变;电针组神经元细胞核基本正常,少数神经细胞可见线粒体轻度肿胀,正常组和假手术组神经细胞则无上述变化,神经细胞胞膜完整,胞浆基质电子密度均匀,生物膜完整,结构清楚,未见明显病变。结论本实验的结果表明:Meynert核注射微量凝聚态Aβ_1-40可诱发CNS的免疫炎性反应。电针百会、太溪、足三里可减少额叶与海马区小胶质细胞和星形胶质细胞的活化,下调IL-1β与TNF-α、IL-1βmRNA与TNF-αmRNA的表达及P₃₈MAPK的磷酸化,从而阻抑额叶与海马区炎性反应,拮抗Aβ_1-40对CNS的毒性作用,抑制神经元变性、坏死。更多还原

【Abstract】 Objective To observe the immunological inflammatory reaction, phosphorylation of P₃₈MAPK,and neuronic ultrastructural change in frontal lobe and hippocampus of Alzheimer’s disease（AD） rats,and the effect of electroacupuncture on them.And to explore the possible mechanism of treating AD by electroacupuncture.Methods 103 normal SD rats were selected out from 110 rats with 24 months by the method of Y type water maze.Then from the 103 rats,30 rats were randomly selected into normal group and sham-operation group,with 15 rats in each group.The model of Alzheimer disease was established by stereotaxis injection agglutinated Aβ_1-40 into rat’s bilateral Meynert basal nuclei.On the thirty-first day after model preparation,the rats were screened again with the method of Y type water maze.In 15 tests,if the correct times lower 1/3 than that before,the model was the successful model.The model rats were randomly selected into model group and（EA） group,with 16 rats in model group and 17rats in EA group.The points of "Baihui"、"Taixi" and "Zusanli" were selected to treat the rats in EA group by electroacupuncture. The treatment was once a day and 6 days was a course of treatment.The next time of therapy continued after one day’s rest,and the total treatment time was 2 courses.The learning and memory ability was tested in the ethology experiment by Morris water maze,the expression change of microglia、astrocytes,IL-1βand TNF-αof frontal lobe and hippocampus in rats were observed by immunohistochemical method of S ABC,the expression of IL-1βmRNA and TNF-αmRNA were observed by RT-PCR,the phosphorylation of P₃₈MAPK were observed by Western-blot,the neuronic ultrastructural change of frontal lobe and hippocampus in rats were observed by transmission electron microscope.All data were put into the computer and analyzed by SPSS13.0 statistical package.The experiment results were expressed as mean±standard deviation（（?） x±s）.Results（1） To indicate by the results in the ethology experiment by Morris water maze:In the location see trial,on the first day,the escape latent period in model group and EA group was remarkably longer than that in normal group and sham-operation group（P<0.01）;there was no significant difference between model group and EA group,normal group and sham-operation group（P>0.05）;on the second day、third day、fourth day and fifth day,the escape latent period in EA group was obviously shorter than model group（P<0.01）.And the escape latent period shortened gradually in each group as the swimming days increased,but there were obviously difference between them.In space search test,time percentage and frequency percentage of spanning the platform quadrant remarkably decreased,10% percentage and 30%percentage remarkably increased in the rats of model group,there were significant differences compared with normal group and sham-operation group（P<0.01）;time percentage and frequency percentage in EA group remarkably increased,10%percentage and 30%percentage in EA group evidently decreased,there were significant differences compared with model group（P<0.01）.（2） The number of positive cells of GFAP and CD68, and the expression of IL-1βand TNF-αin model group obviously increased, their dyeing was deepened,there were significant differences in comparison with normal group and sham-operation group（P<0.01）;After the treatment of electroacupuncture,they remarkably decreased,there were significant differences in comparison with model group,P<0.01.（3） To sign in the experiment of RT-PCR:the expression of IL-1βmRNA and TNF-αmRNA in frontal lobe and hippocampus of model group evidently enhanced,there were significant differences in comparison with normal group and sham-operation group（P<0.05-0.01）;the expression of IL-βmRNA and TNF-αmRNA in EA group obviously reduced,compareing with model group,P< 0.05-0.01.（4） To show in the experiment of Western-blot:There were expression of p-P₃₈MAPK in frontal lobe and hippocampus of each group,but the expression in model group was maximum,there were significant differences in comparison with normal group and sham-operation group（P< 0.01）;After the treatment of electroacupuncture,the expression remarkably decreased,there were significant differences in comparison with model group（P<0.01）.（5） in frontal lobe and hippocampus of the rats in model group, we observed some nerve cells degenerated by transmission electron microscope,endoplasmic reticulum of inter organelle expanded and mitochondriom swelled.However,in frontal lobe and hippocampus of rats in EA group,cell nucleus was almost normal and mitochondrion in organelle swelled a little.There weren’t above-mentioned changes in the nerve cells of frontal lobe and hippocampus of the rats in normal group and sham-operation group.The cell membrane was integral,electron density in ground substance of cytoplasm was well-distributed,biomembrane was integral and the structure was clear.There weren’t obvious pathological changes in the nerve cells of the rats in these two groups.Conclusion The results in present research indicated that agglutinated Aβ_1-40 injected into rat’s bilateral Meynert basal nuclei could cause the immunological inflammatory reaction in central nervous system. Electroacupuncture can decrease the number of microglial cells vivid and astrocytes vivid,reduce the expression of IL-1βand TNF-α、IL-1βmRNA and TNF-αmRNA、p-P₃₈MAPK,thereby restrain inflammatory reaction in frontal lobe and hippocampus of AD rats,antagonize toxic action in central nervous system caused by ABBBBBBB_1-40,inhibitory nerve cells to degenerate and necrosis.更多还原