【作者】 周华；

【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】 目的建立AD大鼠模型,探讨针灸预处理对老年性痴呆模型大鼠Wnt₁信号转导通路的影响,揭示针灸防治AD的机制。方法在大鼠双侧海马区注射Aβ_25-35,建立AD大鼠模型,分别随机分为正常组、假手术组、模型组、预艾灸组、预电针组、预电针加灸组,一共6组,每组各20只。通过预电针和预艾灸大鼠的百会和肾俞穴28天后,观察各组大鼠wnt信号转导相关指标的变化。通过Morris水迷宫检测行为学,免疫组化和免疫印迹检测大鼠海马组织轴蛋白和β-连环蛋白,通过酶联免疫法检测蛋白质磷酸酶2A和糖原合酶激酶-3β的活性,通过实时定量PCR技术检测Wnt1基因表达,并通过超微电镜观察海马组织超微病理变化。结果1.水迷宫检测行为学发现模型组大鼠学习记忆能力有明显损害,其平均潜伏期、经过中心区域的比例、平均游过的路径距离与正常组和假手术组有显著差异（p<0.01）,而预电针和预艾灸对其有明显的预防和改善作用,其中以预电针加灸的疗效最好。2.通过免疫组化和免疫印迹观察到模型组大鼠海马组织轴蛋白表达明显上调,β-连环蛋白表达明显下调,而预艾灸、预电针、预电针加灸对此具有不同程度的调节作用。3.通过酶联免疫法观察到模型组GSK-3β的活性比正常组亦明显升高（p<0.01）,而PP2A的活性比正常组和假手术组明显降低。预电针和预艾灸均可起到调节作用。4.病理组织切片观察到AD大鼠脑组织海马区神经元均有不同程度的破坏,预电针和预艾灸对海马区神经元的损伤具有明显的保护作用。5.通过实时定量PCR技术检测Wnt1基因表达,观察到模型组大鼠海马Wnt1信号异常减弱,与正常组和假手术组比较有显著性差异（p<0.01）。而预艾灸组、预电针组、预电针加灸组大鼠海马Wnt1的信号均有不同程度的增强。结论1.老年性痴呆的基本病机为髓海不足、神机失用,针灸治疗的法则为益精填髓,醒神开窍。百会和肾俞可作为防治该病的首选要穴。2.预电针和预艾灸对于AD模型大鼠的神经功能具有明显的保护作用,有效改善AD模型大鼠学习和记忆功能,减少神经元的损伤,以预电针和预艾灸联合运用疗效最佳。3.预电针和预艾灸能明显下调AD模型大鼠海马组织Axin表达,上调β-连环蛋白表达,降低GSK-3β的活性,升高PP2A的活性,增强Wnt1基因表达。4.针灸防治老年性痴呆的作用机制可能是通过调节Wnt信号转导通路,影响了学习记忆相关酶及蛋白在海马区的表达而发挥作用的。更多还原

【Abstract】 ObjectiveTo establish a rat model of AD to explore the pre-disposal on the rat model of senile dementia Wnt signal transduction pathway,and reveal the mechanism of acupuncture treatment of AD.MethodsAβ_25-35 is injected into the bilateral hippocampal zones of rats to establish the AD model.All the rats were divided into six groups at random,and sham group,the model group,per-moxibustion group,per- electro-acupuncture groups,per-electro-acupuncture and moxibustion group,each group has 20 rats.after the curative by per- electro-acupuncture and moxibustion rats’ baihui and shenshu points for 28 days,the observation group rats Wnt signal transduction related indexes of change.Through the Morris water labyrinth to detecte the change of the rats’ behavior,immunohistochemical and immuno blotting test to inspecte the organization Axin andβ-Catenin of the rats’ hippocamp,ELISA method to detect the activity of PP2A and GSK-3β, real-time quantitatie polymerase chain reaction（PCR） technology to test the Wnt1 gene expression,ultrastructural electron microscope to observe the pathological changes of all the rats’ hippocampal organization.Results1.Morris water labyrinth test found model rats’ ability of learning and memory had been damaged obviously.the average of preclinical period,the ratio of acrossing the center area,average swimming distance of acrossing the path have significant difference between the normal group and sham group（p < 0.01）,per-moxibustion and per-electro -acupuncture have obvious prevention and improvement effect,per-electro-acupuncture and moxibustion has the greatest effect. 2.By immunohistochemical and immuno blotting test,we can observe that the model rats’ hippocampal organization axin of protein expression obviously up-regulated andβ-Catenin protein expression down-regulated obviously, while per-electro-acupuncture,per- moxibustion and per-electro-acupuncture and moxibustion all can change that in different degrees.3.Through ELISA method to observe that the activity of GSK-3βin model group has been increased significantly than normal group also（p < 0.01）,but activity of PP2A has been decreased obviously than normal and sham groups.per-electro-acupuncture and per-moxibustion can both adjust that changes.4.The pathologic biopsy observed AD rats’ hippocampus neurons are damaged in different degree.per-electro-acupuncture and per- moxibustion has obvious protection to that damage.5.Through real-time quantitatie polymerase chain reaction（PCR） technology,Wnt1 gene singal expression was observed to weaken abnormally in model rats.There are significant difference between the model group and sham groups and normal group（p < 0.01）.while the Wntl singal are reinforced in the per-electro-acupuncture group,per- moxibustion group and per-electro-acupuncture group in different degree.Conclusions1.The basic pathogenesis of senile dementia is the marrow of brain is absent and the spirit key can not be used.the treatment law of acupuncture is to tonify the kidney essence and fill up the marrow of brain.inducing resuscitation and open the key to the mind.shenshu and baihui point can be used to prevent and cure the disease for the first choice.2.per-electro-acupuncture and per-moxibustion has obvious protection effect to the AD model rats’ neural function,which can effectively improve the learning and memory function of AD rat models,reduce the damage nerve cell,and the combination of the per- electro-acupuncture and per-moxibustion has the greatest effect to it.3.Per-electro-acupuncture and per-moxibustion can obviously downegulate axin protein expression in hippocampal tissue,up-regulateβ-Catenin protein expression,reduce GSK3βand increase PP2A activity,and reduce the activity Wnt1 gene expression.4.The adjusting mechanism of acupuncture to prevente and cure dementia is likely to regulare the Wnt signal transduction pathways and affect the relevant enzymes and proteins’ expression of learning and memory in the hippocampus.更多还原