天泰Ⅱ号方抗大鼠阿尔茨海默病作用及其机制的实验研究
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摘要
Alzheimer病(AD)是发生在中老年期的一种进行性、不可逆性中枢神经退行性变性疾病，其临床表现为记忆力进行性减退、学习困难、运算障碍、失认失用、甚至生活无法自理等。随着全球人口的老龄化，AD已经成为威胁老年人健康的第四大杀手。全球估计有1500万人患此病，而这个数目还在上升。该病不仅严重影响老年人的身心健康与生活质量，还给家庭和社会带来沉重的负担，已引起全世界的普遍关注。我国已经步入老龄大国，加强AD的研究对我国而言，不仅具有重大的现实意义，而且具有深远的历史影响。
AD主要的神经病理改变有：神经元丧失、神经元纤维缠结(NFT)、大脑皮质和某些皮质下结构出现老年斑(SP)或称神经炎斑(Neuriticplaques，NP)、颗粒空泡变性(granulovacuolar degeneration，GD)、神经毡细丝(Neuropil Threads，NT)和淀粉样血管病(amyloidangiopathy，AAP)等。这此病理改变之间的相互关系还不甚清楚，有研究认为AD发病的生物化学基础是细胞外的淀粉样蛋白(Aβ)沉着和细胞内的微管相关蛋白——Tau蛋白过度磷酸化相互影响的结果。
目前尚无有效的AD治疗药物，减少AD发病以预防为主。有资料表明大多数的AD病例是由MCI(轻度认知障碍)发展而来，既然缺乏肯定的治疗方法和药物，利用中医药治未病的优势进行AD针对性的早期干预应该是有效的策略。导师吴正治研究员一直从事老年脑病的中医药研究，筛选古今验方结合自己的临床开发出天泰Ⅱ号方，进行抗MCI和AD的药理学相关研究。本人在导师过去研究成果的基础上主要进行该方抗AD的药效学及其可能的机制研究。
本课题研究包括两大部分：一是动物实验研究。二是细胞生物学实验研究。
一、动物实验研究
先用Morris水迷宫实验进行动物筛选，将定位航行时间超过平均值30％的大鼠剔除，剩余大鼠供实验用。
大鼠AD疾病模型制备。按1．5ml／kg体重腹腔注射3％的戊巴比妥钠水溶液麻醉大鼠，将麻醉大鼠俯卧位固定在脑立体定位仪上，常规打开鼠头顶部的皮肤，以前囟后3．6mm中线旁开2．5mm为颅骨标志点，用牙科钻在左右两个标志点各钻一孔，将“老化”的1μl 5mM Aβ_(25-35)用微量注射器在5分钟内注入颅骨骨膜下3．0mm海马组织并留针10分钟，术后常规处理。第26天开始Morris水迷宫实验，连续实验5天，将行为学实验结果进行统计学分析。除正常对照组动物颈背部皮下注射0．9％生理盐水0．6ml／kg体重外，AD模型组、假手术组大鼠均皮下注射25％D-半乳糖150mg／kg体重。末次行为学实验结束后断头处死大鼠，取左半脑用4％多聚甲醛固定，进行常规HE染色，观察脑组织结构、细胞的形态和海马CA2、CA3区神经元数量，根据行为学和组织学实验结果评价AD模型的成功与否。
在制备大鼠AD疾病模型成功的基础上，按以上方法制备大鼠AD疾病模型。将大鼠随机分为7组，即天泰Ⅱ号方大、中、小剂量3个组，单味药(№1药)组，AD模型组与正常对照组和假手术组，每组10只大鼠(SD大鼠、SPF级、雄性、240-27 0g，均饲养在屏障环境中，常规给食物和水)，天泰Ⅱ号方大、中、小剂量组大鼠分别灌胃给予1 0％、5％、2．5％的天泰Ⅱ号方10ml／kg，单味药(导师命名为№1药，下同)组灌胃给予0．5％的№1药10ml／kg，假手术组和正常对照组则以相同的方式给予同体积的生理盐水，每日1次，连续用药3 0天。
从给药(天泰Ⅱ号方)第26天开始进行行为学实验，实验内容包括学习训练、定位航行实验和空间探索实验三部分。末次行为学实验结束后断头处死大鼠，取全脑。其右脑保存于液氮中，左脑用4％多聚甲醛固定。左脑进行常规HE染色，观察脑组织结构、细胞的形态和海马CA2、CA3区神经元数量；用免疫组织化学方法(SP法)研究大鼠前脑抗ChAT抗体阳性细胞数量的变化；用TUNEL法研究脑组织神经细胞凋亡的情况，凋亡指数。
用真核细胞裂解液裂解右脑组织20min抽提脑组织总蛋白，在4℃以12000rpm离心10分钟，获取的上清即为含目的蛋白的混合物。使用蛋白质印迹实验方法，经电泳、转膜和免疫反应、化学发光显示CHOP蛋白，用凝胶图像分析仪检测。
另外，从假手术组、AD模型组、天泰Ⅱ号方大剂量组和单味药(№1药)组中每组随机取脑组织3个，用Unizol抽提脑组织总RNA，经检测每个样本RNA的OD_(260)／OD_(280)值均大于等于1．86，RNA电泳5S、18S、28S条带清晰，经电泳和浓度检测符合实验要求。按照Illumina的RNA扩增试剂盒的方法以RNA为模板合成cDNA，以纯化的cDNA为模板合成cRNA，经过Cy3荧光标记后与RatRef-12芯片杂交，用BeadSation进行扫描并用软件分析大鼠脑组织表达谱基因是否存在差异。
二、细胞生物学实验。
首先制备载药脑脊液。将家兔(清洁级、2．4-2．7kg、雌雄各半)随机分为3组，即正常组、天泰Ⅱ号方组、单味药组，每组6只，常规饲养。天泰Ⅱ号方组灌胃给予家兔20％天泰Ⅱ号方10ml／kg，单味药组灌胃给予1％№l药10ml／kg。每天2次，共7天。末次给药1小时后颈部放血处死家兔，用穿刺针刺入小脑延髓池抽取脑脊液800-1000μl。将同组各家兔脑脊液混合后4℃12000rpm离心10分钟，取上清经孔径0．22μm的滤器过滤除菌，即得载药CSF，-70℃保存备用。
胎鼠神经干细胞的原代培养与转化。用神经球悬浮法(培养液为DMEM／F12、20ng／ml EGF、20ng／ml bFGF)培养神经干细胞(Neural stem cell，NSC)，将单克隆传代至第三代后用含胎牛血清(Fetal bovin serum，FBS)的NSC转化液处理NSC，后者分化为形态各异的多种细胞，用免疫细胞化学(SP法)和免疫细胞荧光行抗NSE、抗Nestin、抗GFAP、抗GC、抗ox42鉴定。经鉴定培养的是NSC后，NSC培养液中加入载药CSF，观察NSC的生长情况；NSC转化液中加入载药(天泰Ⅱ号方)脑脊液后观察NSC的转化情况。使用鸡胚腿骨骼肌提取液、载药CSF、自然转化液观察NSC向胆碱能神经元的转化率。
胎鼠海马神经元的原代培养和载药(天泰Ⅱ号方)脑脊液对AD细胞模型的影响。以Neurobasal A、2％B-2 7、谷氨酰胺和2-Me混合液为神经元的培养液进行无血清培养，经7-8d培养出大量的细胞，用免疫细胞化学法行抗NSE鉴定，鉴定为神经元后用于后续实验。
神经元原代培养7天左右待神经元长到对数期时向培养液中加入终浓度为10μM的“老化”的A[_(25-35)孵育24小时，用MTT法分析细胞生长能力；行吖啶橙-溴化乙锭染色，荧光显微镜观察细胞凋亡情况，统计学分析结果，评价AD细胞模型。对成功的AD细胞模型用载药脑脊液进行干预，用MTT法分析细胞生长能力；并行吖啶橙-溴化乙锭染色，荧光显微镜观察细胞情况，计细胞凋亡数，计算凋亡指数，统计学分析结果。
结果：(一)、经过Morris水迷宫实验和脑组织HE染色观察、比较海马CA2、CA3的神经元数量，认为海马双侧注射Aβ_(25-35)合用D-半乳糖制造的AD模型是成功的；(二)、通过Morris水迷宫测试，与AD模型组相比，天泰Ⅱ号方大、中、小剂量组和单味药组大鼠在学习训练、定位航行、空间探索实验时到达安全平台的时间和距离均缩短，在空间探索实验时大鼠穿越安全平台次数增加，提示天泰Ⅱ号方能明显改善AD大鼠的学习记忆能力。虽然天泰Ⅱ号方大、中、小剂量组比较没有显著地统计学差异，但从数值上看大剂量略有优势；(三)取左半脑组织的石蜡块作3μm厚切片，经HE染色观察发现，AD模型组海马神经细胞排列紊乱、层次减少，可见核固缩现象；CA2、CA3区神经元数量明显少于天泰Ⅱ号方大、中、小剂量组和单味药组。切片经常规脱蜡水化处理后行抗ChAT免疫组化染色(SP法)研究，在端脑区可见AD组阳性细胞数量明显少于天泰Ⅱ号方大、中、小剂量组(P＜0．05)；(四)在左半脑组织TUNEL法实验中发现，AD组细胞凋亡明显，天泰Ⅱ号方大剂量组中枢神经细胞凋亡显著少于其他组，凋亡指数较低；(五)从右脑组织抽提的总蛋白，使用蛋白质印迹实验方法，经电泳、转膜和免疫反应、化学发光显示CHOP蛋白，用凝胶图像分析仪检测发现CHOP蛋白表达少于其他组，存在明显差异(P＜0．05)；(六)用BeadSation进行杂交芯片扫描并通过专业软件分析，不同组之间进行两两比较发现：AD模型组对假手术组比较有39个基因存在差异表达；AD模型组对天泰2号方大剂量组比较有107个基因存在差异表达；AD模型组对单味药组比较有56个基因存在差异表达；天泰2号方型组对假手术组比较有38个基因存在差异表达；单味药型组对假手术组比较有102个基因存在差异表达；天泰2号方型对单味药组比较有204个基因存在差异表达。
(七)“神经球”和分化出的形态各异的多种细胞，经鉴定为NSC、星形胶质细胞、少突胶质细胞、神经元等，我们认为用“神经球”悬浮法培养NSC成功；(八)向NSC培养液中加入载药CSF后NSC的生长加快；NSC的自然分化液中加入载药(天泰Ⅱ号方)脑脊液使NSC的转化率提高。鸡胚腿骨骼肌提取液可提高NSC向胆碱能神经元的转化率。加入载药(天泰Ⅱ号方)脑脊液使NSC向胆碱能神经元的定向转化率增高。(九)取白海马的细胞经无血清培养，7-8d后可以进入对数生长期，细胞经抗NSE免疫细胞化学鉴定为神经元。待神经元长到对数期时向培养液中加入终浓度为10μM的“老化”的Aβ_(25-35)孵育24小时，神经细胞生长受到抑制，细胞形态变小并出现大量细胞凋亡，认为AD细胞模型制造成功。对AD细胞模型用载药脑脊液进行干预，用MTT法分析细胞生长能力；并行吖啶橙-溴化乙锭染色，荧光显微镜下细胞计数，计算凋亡指数，统计学分析结果表明天泰Ⅱ号方的抗AD作用。
综上所述，天泰Ⅱ号方有较好的抗大鼠AD作用，其机理涉及抗凋亡、抗内质网应激、促进NSC的分化、转化，尤其是向胆碱能神经的转化有关。
Alzheimer disease(AD)is a progressive disease which the degeneration of the central nervous system is irreversible.It occurs in aging elders and its clinical manifestations include progressively memory declining、learning difficultly,dyscalculia,apraxia,agnosia,or even the AD patients can not take care of themselves and so on.the German doctor,Alois Alzheimer,first reported the clinical cases.the clinical reports such as Alzheimer diseases have been rapidly increasing Since 1907.With the global aging of the population,AD has became a fourth largestwhich threat to the health of the elders.
Major neuropathological change of Alzheimer disease includes:lossof neurons;neurofibrillary tangles(NFT)and some senile plaques(SP),or neuritic plaques(NP)in the cerebral cortex and subcortical structure,which are responsible for higher brain functions;granulovacuolar degeneration(GD);neuropil threads(NT)and amyloid angiopathy(AAP)and so on.The inter-relationship among these pathological changes has been uncertain.The plaques are deposits found outside the neurons and are composed primarily of a small protein called amyloid-beta,or A-beta.The tangles are located inside neurons and their branching projections(axons and dendrites)and are made of filaments of a protein calledtau.Are the plaques and tangles responsible for the degeneration of brain neurons,or are they merely markers of where neuronal death has already occurred ? In the past decades,the weight of evidence has shifted toward the amyloid-cascade hypothesis,which posits that both A-betaand tau are intimately involved in causing Alzheimer's disease,with A-beta providing the initial insult.A-beta molecules in a test tube can assemble into fiberlike structures similar to those found in the plaques of Alzheimer's disease.The soluble assemblies as well as the fibers of A-beta are toxic to neurons cultured in Petri dishes,and the former can interfere with processes critical to learning and memory in mice.
As we known that there is no effective treatment and drugs for AD now.It is the idealest method to prevent generateion and progress ofAD.Research datas show that the majority of AD cases are progressedfrom MCI(mild cognitive impairment)case.althoug it is lack of the definitive treatment and drugs on AD,Chinese traditional medicine is good at against the early stage of disease and Subhealth.
Professor Wu has researched and practiced on the elderly brain diseases treated with Chinese traditional medicine for a long time He developed his own compound recipe TIANTAIⅡon Alzheimer's disease ,screening a lot of ancient and modern compound recipe on AD andcombing with the his clinical practices.It has been studying on anti-MCI and AD.On the basis of past researches about its pharmacodynamic effects I continue the study on AD rats and hope to discove the major effects and its possible mechanism.
My research includes two major parts:The first experiment was studied on AD rats.The second one is study on primary stem cells and neurons.SectionⅠ.Experimental study of the protective effect and its mechanismof TIANTAI II on AD rats
First,Morris water maze was used for screening rats,which wereSD rats、SPF-class、male、240-270g weight,were rearing in barrier environment and seeding the conventional food and water.They were removed whose journey time was more than 30% to the average,the re-maining rats were used for experiments.
The rats were anesthetized by intraperitoneal injection with 3% aqueous solution of sodium pentobarbital 1.5ml/kg weight.Then those ratswere pronely fixed in the brain three-dimensional positioning instruments.At first we conventionally open the skin at the top of the head,themarker point was beside 2.5mm and behind 3.6mm to the Bregma.Wedrilled two holes at the marker point with the special dental drill andinserted the microsyringes into the hippocampus 3.0mm deepth under the parietal bone.1μl of 5mM“aging”Aβ_(25-35)was slowly injected into the hippocampus in 5 minutes and the microsyringes were left for 10 minutes After that,the injured skins were carefully sutured and the ratswere treated by intramuscular injected 80 thousand unit of Penicillin for3days.
From the 26th day after the operation,Morris water maze experiment was began,the experiment lasted for 5 days.the experimental resuIts of ability of study and memory were analyzed statistically.In addition to the normal control group of rats was subcutaneous injected 0.6ml/kg of 0.9% normal saline solution in the back and neck outside each rat,AD model group,sham-operated rats were subcutaneous injected 150mg/kg of 25% D-galactose each rat.At the end of the behavior experiment all rats were killed through the decapitation.The left brains were fixed with 4% paraformaldehyde for 24 hours,and were regularly stained by HE stain and we observed the morphology of organizational structureand neurons in the CA2、CA3 area of hippocampal We evaluate the AD model in accordance with the behavioral results and the histological results.
The AD model rats were randomly divided into 5 groups,TIANTAⅠⅡ3 dose groups(10%,5%,2.5%),single drug(№1 drug)group andAD model group,while there was the establishment of normal controlgroup and the sham-operated group,10 rats in each group.TIANTAIⅡlarge-dose group rats were given oral 10%,and TIANTAIⅡmedium-dose group rats were given oral 5% and TIANTAIⅡsmall-dose grouprats were given oral 2.5% of TIANTAIⅡ,the volume was 10ml per 1kg weight.Single drug(№1 drug)group given oral the same volumeof 0.5%№1 drug.the rats of the sham-operated group and the normal control group were given the same volume of normal saline,the manner was same as the others The administration was once a day for30 days continuously.all rats except the normal group were subcutaneous injected 150mg/kg of 25% D-galactose each rat for 50 days.
The behavioral experiments were stared from the 26th day of the administration,including the training of study,locational navigation and3D-space exploration At the end of the behavior experiment all ratswere killed through the decapitation.The left brains were fixed with 4%paraformaldehyde for 24 hours,and were regularly stained by HE stainand we observed the morphology of organizational structure and neurons in the CA2、CA3 area of hippocampal We evaluate the AD model in accordance with the behavioral results and the histological results We studied the morphology and number value of the cholinergic neuron in telencephalon by immunohistochemistry,we studied the changes of thepositive cells of anti-ChAT antibody And we studied the neuronal apoptosis in brain tissue by TUNEL method The right brain was storedin liquid nitrogen for studing the expression of CHOP protein in the brains.by Western Blotting.
Three brain samples were randomly selected each group,and we extracted the total RNA from each sample brain The RNA samples were tested,the value of the rato OD_(260)/OD_(280) were over 1.86 and met the experimental requires.We synthesized and amplified cDNA and hybridizated with the RatRef-12 gene chip.And we scaned the genic chipand analysis the difference of the genic expression of 12 rat brains
Compared the AD model group with the others,the distance and the positioning time were shorter And the distance and time of locatioal navigation were shorter,the distance of the space exploration was shorter and the times crossing the security platform increased.IIDoes-large group and dose-medium group of TIANTAI had conspicuously no difference TIANTAI II can significantly improve the ability of learningand memory of AD rats.
Experimental study of HE staining and anti-ChAT mmunohistochemistry on the brains shows that the number of the neurons in CA2、CA3area of hippocampal of TIANTAIⅡgroups(large dose and medium dose)were more than one of AD rats.And the number of cholinergicneurons in forebrain was more than the multi-AD model group(P＜0.05)
Expression of CHOP was less than AD model group.
And apoptosis neurons was less than AD group.We scaned the genic chip and analysis the difference of the genic expression among ADmodel group,TIANTAIⅡgroup(large dose),single herb(№1)groupand sham-operated group We found a lot of different expression of the gene.
SectionⅡ.Experimental study of the effect of TIANTAIⅡ-containingcerebrospinal fluid.on primary cultured neural stem cells and neurons
TIANTAIⅡ-containing cerebrospinal fluid was made for future cel1 biology experiments.18 Rabbits(Clean class,2.4-2.7 kg weight,malewere half.Female was half)were randomly divided into 3 groups There were the normal group,TIANTAIⅡgroup and single drug(№1)groups.Each group had 6 rabbits.Each rabbit of TiantaiⅡGroup wasgiven oral 10ml/kg of 20% TIANTAIⅡfor 7days,twice a day.
An hour after the end of administration,the rabbits were killed bybleeding to death We have got 800-1000μl of TIANTAIⅡ-containing cerebrospinal fluid each rabbit with the microsyringe from fourth ventricle through the foramen magnum.The CSF samples of the same group were mixed and centrifugated 12000rpm for 10 minutes at 4℃.The supernatant from the CSF were sterilized with经孔径0.22μm filter and reserved the bacteriafree CSF containing TIANTAIⅡCSF at-70℃Cfor a long time.
Neural stem cells(NSC)from fetal rats were primary cultured andtransformated Neurospheres formation:a single suspension of EB weregot by trypsinization for 5 minutes with 0.25% trypsin in 1 mmol/L EDTA(GIBCO/Invitrogen,USA),followed by quenching with 10% FCS.The cells were washed twice with basic neurosphere medium composed of DMEM:F12(1:1),0.6% glucose,2 mmol/L glutamine,3 M sodium bicarbonate,5 mmol/L HEPES buffer,25μg/ml insulin,100μg/ml transferring,20 nmol/L progesterone,60μmol/L putrescine,30 nmol/L selenium chloride [all from Sigma(USA)except glutamine(sigma)from GIBCO/Invitrogen(USA)] and plated onto T75 flasks(Greiner,Germany)at a density of 1×10(5)cells/ml in the neurosphere medium(basic neurosphere medium supplemented with 20 ng/ml fibroblast growth factor-2)(Invitrogen,USA)and 2μg/ml heparin sulfate(Sigma,USA).Cellswere grown for five to seven days and formed neurospheres.NSC were cultured with 10% fetal bovine serum(FBS)and could differentiate into astrocytes,oligodendrocyte cells,neurons,etc.And we found NSC differentiate into Cholinergic neurons incubated with the extract from feta1 chick leg muscles and TIANTAIⅡ-containing CSF could promote theNSC into cholinergic neurons.
The neurons from the fetal rat(E16-18)hippocampus were culturedwith the medium(includes Neurobasl A medium,2-Mercaptoethanol,EGF,bFGF,Glutamine,penicillin,stepmycin)at 37℃5%CO_2.Thecultured cells were identified as neurons and were used for the followthe experiment.When neurons grew to the logarithmic phase and were incubated with 10μM of Aβ_(25-35)for 24-hour and the AD cell modelwas made And Aβ_(25-35)caused neurons apoptosis and injured MTT method was used for analysis of neurons' growth capacity and AD model.neurons were grown well with TIANTAIⅡ-containing cerebrospinal fluid.With acridine orange-ethidium bromide staining,we counted the value of neirons 1 under fluorescence microscope and calculated the apoptotic index.All results were statistical analyzed.
To sum up,TIANTAI II has a better role in anti-AD of rats,themechanism involves anti-apoptosis,anti-endoplasmic reticulum stress,andpromote the differentiation and transformation of NSC And It can promote the NSC differentiate to cholinergic neurons in particular.Thereis whether or not occurrence of differentially expressed genes,we hopethe further studies on TIANTAIⅡ.
AD主要的神经病理改变有：神经元丧失、神经元纤维缠结(NFT)、大脑皮质和某些皮质下结构出现老年斑(SP)或称神经炎斑(Neuriticplaques，NP)、颗粒空泡变性(granulovacuolar degeneration，GD)、神经毡细丝(Neuropil Threads，NT)和淀粉样血管病(amyloidangiopathy，AAP)等。这此病理改变之间的相互关系还不甚清楚，有研究认为AD发病的生物化学基础是细胞外的淀粉样蛋白(Aβ)沉着和细胞内的微管相关蛋白——Tau蛋白过度磷酸化相互影响的结果。
目前尚无有效的AD治疗药物，减少AD发病以预防为主。有资料表明大多数的AD病例是由MCI(轻度认知障碍)发展而来，既然缺乏肯定的治疗方法和药物，利用中医药治未病的优势进行AD针对性的早期干预应该是有效的策略。导师吴正治研究员一直从事老年脑病的中医药研究，筛选古今验方结合自己的临床开发出天泰Ⅱ号方，进行抗MCI和AD的药理学相关研究。本人在导师过去研究成果的基础上主要进行该方抗AD的药效学及其可能的机制研究。
本课题研究包括两大部分：一是动物实验研究。二是细胞生物学实验研究。
一、动物实验研究
先用Morris水迷宫实验进行动物筛选，将定位航行时间超过平均值30％的大鼠剔除，剩余大鼠供实验用。
大鼠AD疾病模型制备。按1．5ml／kg体重腹腔注射3％的戊巴比妥钠水溶液麻醉大鼠，将麻醉大鼠俯卧位固定在脑立体定位仪上，常规打开鼠头顶部的皮肤，以前囟后3．6mm中线旁开2．5mm为颅骨标志点，用牙科钻在左右两个标志点各钻一孔，将“老化”的1μl 5mM Aβ_(25-35)用微量注射器在5分钟内注入颅骨骨膜下3．0mm海马组织并留针10分钟，术后常规处理。第26天开始Morris水迷宫实验，连续实验5天，将行为学实验结果进行统计学分析。除正常对照组动物颈背部皮下注射0．9％生理盐水0．6ml／kg体重外，AD模型组、假手术组大鼠均皮下注射25％D-半乳糖150mg／kg体重。末次行为学实验结束后断头处死大鼠，取左半脑用4％多聚甲醛固定，进行常规HE染色，观察脑组织结构、细胞的形态和海马CA2、CA3区神经元数量，根据行为学和组织学实验结果评价AD模型的成功与否。
在制备大鼠AD疾病模型成功的基础上，按以上方法制备大鼠AD疾病模型。将大鼠随机分为7组，即天泰Ⅱ号方大、中、小剂量3个组，单味药(№1药)组，AD模型组与正常对照组和假手术组，每组10只大鼠(SD大鼠、SPF级、雄性、240-27 0g，均饲养在屏障环境中，常规给食物和水)，天泰Ⅱ号方大、中、小剂量组大鼠分别灌胃给予1 0％、5％、2．5％的天泰Ⅱ号方10ml／kg，单味药(导师命名为№1药，下同)组灌胃给予0．5％的№1药10ml／kg，假手术组和正常对照组则以相同的方式给予同体积的生理盐水，每日1次，连续用药3 0天。
从给药(天泰Ⅱ号方)第26天开始进行行为学实验，实验内容包括学习训练、定位航行实验和空间探索实验三部分。末次行为学实验结束后断头处死大鼠，取全脑。其右脑保存于液氮中，左脑用4％多聚甲醛固定。左脑进行常规HE染色，观察脑组织结构、细胞的形态和海马CA2、CA3区神经元数量；用免疫组织化学方法(SP法)研究大鼠前脑抗ChAT抗体阳性细胞数量的变化；用TUNEL法研究脑组织神经细胞凋亡的情况，凋亡指数。
用真核细胞裂解液裂解右脑组织20min抽提脑组织总蛋白，在4℃以12000rpm离心10分钟，获取的上清即为含目的蛋白的混合物。使用蛋白质印迹实验方法，经电泳、转膜和免疫反应、化学发光显示CHOP蛋白，用凝胶图像分析仪检测。
另外，从假手术组、AD模型组、天泰Ⅱ号方大剂量组和单味药(№1药)组中每组随机取脑组织3个，用Unizol抽提脑组织总RNA，经检测每个样本RNA的OD_(260)／OD_(280)值均大于等于1．86，RNA电泳5S、18S、28S条带清晰，经电泳和浓度检测符合实验要求。按照Illumina的RNA扩增试剂盒的方法以RNA为模板合成cDNA，以纯化的cDNA为模板合成cRNA，经过Cy3荧光标记后与RatRef-12芯片杂交，用BeadSation进行扫描并用软件分析大鼠脑组织表达谱基因是否存在差异。
二、细胞生物学实验。
首先制备载药脑脊液。将家兔(清洁级、2．4-2．7kg、雌雄各半)随机分为3组，即正常组、天泰Ⅱ号方组、单味药组，每组6只，常规饲养。天泰Ⅱ号方组灌胃给予家兔20％天泰Ⅱ号方10ml／kg，单味药组灌胃给予1％№l药10ml／kg。每天2次，共7天。末次给药1小时后颈部放血处死家兔，用穿刺针刺入小脑延髓池抽取脑脊液800-1000μl。将同组各家兔脑脊液混合后4℃12000rpm离心10分钟，取上清经孔径0．22μm的滤器过滤除菌，即得载药CSF，-70℃保存备用。
胎鼠神经干细胞的原代培养与转化。用神经球悬浮法(培养液为DMEM／F12、20ng／ml EGF、20ng／ml bFGF)培养神经干细胞(Neural stem cell，NSC)，将单克隆传代至第三代后用含胎牛血清(Fetal bovin serum，FBS)的NSC转化液处理NSC，后者分化为形态各异的多种细胞，用免疫细胞化学(SP法)和免疫细胞荧光行抗NSE、抗Nestin、抗GFAP、抗GC、抗ox42鉴定。经鉴定培养的是NSC后，NSC培养液中加入载药CSF，观察NSC的生长情况；NSC转化液中加入载药(天泰Ⅱ号方)脑脊液后观察NSC的转化情况。使用鸡胚腿骨骼肌提取液、载药CSF、自然转化液观察NSC向胆碱能神经元的转化率。
胎鼠海马神经元的原代培养和载药(天泰Ⅱ号方)脑脊液对AD细胞模型的影响。以Neurobasal A、2％B-2 7、谷氨酰胺和2-Me混合液为神经元的培养液进行无血清培养，经7-8d培养出大量的细胞，用免疫细胞化学法行抗NSE鉴定，鉴定为神经元后用于后续实验。
神经元原代培养7天左右待神经元长到对数期时向培养液中加入终浓度为10μM的“老化”的A[_(25-35)孵育24小时，用MTT法分析细胞生长能力；行吖啶橙-溴化乙锭染色，荧光显微镜观察细胞凋亡情况，统计学分析结果，评价AD细胞模型。对成功的AD细胞模型用载药脑脊液进行干预，用MTT法分析细胞生长能力；并行吖啶橙-溴化乙锭染色，荧光显微镜观察细胞情况，计细胞凋亡数，计算凋亡指数，统计学分析结果。
结果：(一)、经过Morris水迷宫实验和脑组织HE染色观察、比较海马CA2、CA3的神经元数量，认为海马双侧注射Aβ_(25-35)合用D-半乳糖制造的AD模型是成功的；(二)、通过Morris水迷宫测试，与AD模型组相比，天泰Ⅱ号方大、中、小剂量组和单味药组大鼠在学习训练、定位航行、空间探索实验时到达安全平台的时间和距离均缩短，在空间探索实验时大鼠穿越安全平台次数增加，提示天泰Ⅱ号方能明显改善AD大鼠的学习记忆能力。虽然天泰Ⅱ号方大、中、小剂量组比较没有显著地统计学差异，但从数值上看大剂量略有优势；(三)取左半脑组织的石蜡块作3μm厚切片，经HE染色观察发现，AD模型组海马神经细胞排列紊乱、层次减少，可见核固缩现象；CA2、CA3区神经元数量明显少于天泰Ⅱ号方大、中、小剂量组和单味药组。切片经常规脱蜡水化处理后行抗ChAT免疫组化染色(SP法)研究，在端脑区可见AD组阳性细胞数量明显少于天泰Ⅱ号方大、中、小剂量组(P＜0．05)；(四)在左半脑组织TUNEL法实验中发现，AD组细胞凋亡明显，天泰Ⅱ号方大剂量组中枢神经细胞凋亡显著少于其他组，凋亡指数较低；(五)从右脑组织抽提的总蛋白，使用蛋白质印迹实验方法，经电泳、转膜和免疫反应、化学发光显示CHOP蛋白，用凝胶图像分析仪检测发现CHOP蛋白表达少于其他组，存在明显差异(P＜0．05)；(六)用BeadSation进行杂交芯片扫描并通过专业软件分析，不同组之间进行两两比较发现：AD模型组对假手术组比较有39个基因存在差异表达；AD模型组对天泰2号方大剂量组比较有107个基因存在差异表达；AD模型组对单味药组比较有56个基因存在差异表达；天泰2号方型组对假手术组比较有38个基因存在差异表达；单味药型组对假手术组比较有102个基因存在差异表达；天泰2号方型对单味药组比较有204个基因存在差异表达。
(七)“神经球”和分化出的形态各异的多种细胞，经鉴定为NSC、星形胶质细胞、少突胶质细胞、神经元等，我们认为用“神经球”悬浮法培养NSC成功；(八)向NSC培养液中加入载药CSF后NSC的生长加快；NSC的自然分化液中加入载药(天泰Ⅱ号方)脑脊液使NSC的转化率提高。鸡胚腿骨骼肌提取液可提高NSC向胆碱能神经元的转化率。加入载药(天泰Ⅱ号方)脑脊液使NSC向胆碱能神经元的定向转化率增高。(九)取白海马的细胞经无血清培养，7-8d后可以进入对数生长期，细胞经抗NSE免疫细胞化学鉴定为神经元。待神经元长到对数期时向培养液中加入终浓度为10μM的“老化”的Aβ_(25-35)孵育24小时，神经细胞生长受到抑制，细胞形态变小并出现大量细胞凋亡，认为AD细胞模型制造成功。对AD细胞模型用载药脑脊液进行干预，用MTT法分析细胞生长能力；并行吖啶橙-溴化乙锭染色，荧光显微镜下细胞计数，计算凋亡指数，统计学分析结果表明天泰Ⅱ号方的抗AD作用。
综上所述，天泰Ⅱ号方有较好的抗大鼠AD作用，其机理涉及抗凋亡、抗内质网应激、促进NSC的分化、转化，尤其是向胆碱能神经的转化有关。
Alzheimer disease(AD)is a progressive disease which the degeneration of the central nervous system is irreversible.It occurs in aging elders and its clinical manifestations include progressively memory declining、learning difficultly,dyscalculia,apraxia,agnosia,or even the AD patients can not take care of themselves and so on.the German doctor,Alois Alzheimer,first reported the clinical cases.the clinical reports such as Alzheimer diseases have been rapidly increasing Since 1907.With the global aging of the population,AD has became a fourth largestwhich threat to the health of the elders.
Major neuropathological change of Alzheimer disease includes:lossof neurons;neurofibrillary tangles(NFT)and some senile plaques(SP),or neuritic plaques(NP)in the cerebral cortex and subcortical structure,which are responsible for higher brain functions;granulovacuolar degeneration(GD);neuropil threads(NT)and amyloid angiopathy(AAP)and so on.The inter-relationship among these pathological changes has been uncertain.The plaques are deposits found outside the neurons and are composed primarily of a small protein called amyloid-beta,or A-beta.The tangles are located inside neurons and their branching projections(axons and dendrites)and are made of filaments of a protein calledtau.Are the plaques and tangles responsible for the degeneration of brain neurons,or are they merely markers of where neuronal death has already occurred ? In the past decades,the weight of evidence has shifted toward the amyloid-cascade hypothesis,which posits that both A-betaand tau are intimately involved in causing Alzheimer's disease,with A-beta providing the initial insult.A-beta molecules in a test tube can assemble into fiberlike structures similar to those found in the plaques of Alzheimer's disease.The soluble assemblies as well as the fibers of A-beta are toxic to neurons cultured in Petri dishes,and the former can interfere with processes critical to learning and memory in mice.
As we known that there is no effective treatment and drugs for AD now.It is the idealest method to prevent generateion and progress ofAD.Research datas show that the majority of AD cases are progressedfrom MCI(mild cognitive impairment)case.althoug it is lack of the definitive treatment and drugs on AD,Chinese traditional medicine is good at against the early stage of disease and Subhealth.
Professor Wu has researched and practiced on the elderly brain diseases treated with Chinese traditional medicine for a long time He developed his own compound recipe TIANTAIⅡon Alzheimer's disease ,screening a lot of ancient and modern compound recipe on AD andcombing with the his clinical practices.It has been studying on anti-MCI and AD.On the basis of past researches about its pharmacodynamic effects I continue the study on AD rats and hope to discove the major effects and its possible mechanism.
My research includes two major parts:The first experiment was studied on AD rats.The second one is study on primary stem cells and neurons.SectionⅠ.Experimental study of the protective effect and its mechanismof TIANTAI II on AD rats
First,Morris water maze was used for screening rats,which wereSD rats、SPF-class、male、240-270g weight,were rearing in barrier environment and seeding the conventional food and water.They were removed whose journey time was more than 30% to the average,the re-maining rats were used for experiments.
The rats were anesthetized by intraperitoneal injection with 3% aqueous solution of sodium pentobarbital 1.5ml/kg weight.Then those ratswere pronely fixed in the brain three-dimensional positioning instruments.At first we conventionally open the skin at the top of the head,themarker point was beside 2.5mm and behind 3.6mm to the Bregma.Wedrilled two holes at the marker point with the special dental drill andinserted the microsyringes into the hippocampus 3.0mm deepth under the parietal bone.1μl of 5mM“aging”Aβ_(25-35)was slowly injected into the hippocampus in 5 minutes and the microsyringes were left for 10 minutes After that,the injured skins were carefully sutured and the ratswere treated by intramuscular injected 80 thousand unit of Penicillin for3days.
From the 26th day after the operation,Morris water maze experiment was began,the experiment lasted for 5 days.the experimental resuIts of ability of study and memory were analyzed statistically.In addition to the normal control group of rats was subcutaneous injected 0.6ml/kg of 0.9% normal saline solution in the back and neck outside each rat,AD model group,sham-operated rats were subcutaneous injected 150mg/kg of 25% D-galactose each rat.At the end of the behavior experiment all rats were killed through the decapitation.The left brains were fixed with 4% paraformaldehyde for 24 hours,and were regularly stained by HE stain and we observed the morphology of organizational structureand neurons in the CA2、CA3 area of hippocampal We evaluate the AD model in accordance with the behavioral results and the histological results.
The AD model rats were randomly divided into 5 groups,TIANTAⅠⅡ3 dose groups(10%,5%,2.5%),single drug(№1 drug)group andAD model group,while there was the establishment of normal controlgroup and the sham-operated group,10 rats in each group.TIANTAIⅡlarge-dose group rats were given oral 10%,and TIANTAIⅡmedium-dose group rats were given oral 5% and TIANTAIⅡsmall-dose grouprats were given oral 2.5% of TIANTAIⅡ,the volume was 10ml per 1kg weight.Single drug(№1 drug)group given oral the same volumeof 0.5%№1 drug.the rats of the sham-operated group and the normal control group were given the same volume of normal saline,the manner was same as the others The administration was once a day for30 days continuously.all rats except the normal group were subcutaneous injected 150mg/kg of 25% D-galactose each rat for 50 days.
The behavioral experiments were stared from the 26th day of the administration,including the training of study,locational navigation and3D-space exploration At the end of the behavior experiment all ratswere killed through the decapitation.The left brains were fixed with 4%paraformaldehyde for 24 hours,and were regularly stained by HE stainand we observed the morphology of organizational structure and neurons in the CA2、CA3 area of hippocampal We evaluate the AD model in accordance with the behavioral results and the histological results We studied the morphology and number value of the cholinergic neuron in telencephalon by immunohistochemistry,we studied the changes of thepositive cells of anti-ChAT antibody And we studied the neuronal apoptosis in brain tissue by TUNEL method The right brain was storedin liquid nitrogen for studing the expression of CHOP protein in the brains.by Western Blotting.
Three brain samples were randomly selected each group,and we extracted the total RNA from each sample brain The RNA samples were tested,the value of the rato OD_(260)/OD_(280) were over 1.86 and met the experimental requires.We synthesized and amplified cDNA and hybridizated with the RatRef-12 gene chip.And we scaned the genic chipand analysis the difference of the genic expression of 12 rat brains
Compared the AD model group with the others,the distance and the positioning time were shorter And the distance and time of locatioal navigation were shorter,the distance of the space exploration was shorter and the times crossing the security platform increased.IIDoes-large group and dose-medium group of TIANTAI had conspicuously no difference TIANTAI II can significantly improve the ability of learningand memory of AD rats.
Experimental study of HE staining and anti-ChAT mmunohistochemistry on the brains shows that the number of the neurons in CA2、CA3area of hippocampal of TIANTAIⅡgroups(large dose and medium dose)were more than one of AD rats.And the number of cholinergicneurons in forebrain was more than the multi-AD model group(P＜0.05)
Expression of CHOP was less than AD model group.
And apoptosis neurons was less than AD group.We scaned the genic chip and analysis the difference of the genic expression among ADmodel group,TIANTAIⅡgroup(large dose),single herb(№1)groupand sham-operated group We found a lot of different expression of the gene.
SectionⅡ.Experimental study of the effect of TIANTAIⅡ-containingcerebrospinal fluid.on primary cultured neural stem cells and neurons
TIANTAIⅡ-containing cerebrospinal fluid was made for future cel1 biology experiments.18 Rabbits(Clean class,2.4-2.7 kg weight,malewere half.Female was half)were randomly divided into 3 groups There were the normal group,TIANTAIⅡgroup and single drug(№1)groups.Each group had 6 rabbits.Each rabbit of TiantaiⅡGroup wasgiven oral 10ml/kg of 20% TIANTAIⅡfor 7days,twice a day.
An hour after the end of administration,the rabbits were killed bybleeding to death We have got 800-1000μl of TIANTAIⅡ-containing cerebrospinal fluid each rabbit with the microsyringe from fourth ventricle through the foramen magnum.The CSF samples of the same group were mixed and centrifugated 12000rpm for 10 minutes at 4℃.The supernatant from the CSF were sterilized with经孔径0.22μm filter and reserved the bacteriafree CSF containing TIANTAIⅡCSF at-70℃Cfor a long time.
Neural stem cells(NSC)from fetal rats were primary cultured andtransformated Neurospheres formation:a single suspension of EB weregot by trypsinization for 5 minutes with 0.25% trypsin in 1 mmol/L EDTA(GIBCO/Invitrogen,USA),followed by quenching with 10% FCS.The cells were washed twice with basic neurosphere medium composed of DMEM:F12(1:1),0.6% glucose,2 mmol/L glutamine,3 M sodium bicarbonate,5 mmol/L HEPES buffer,25μg/ml insulin,100μg/ml transferring,20 nmol/L progesterone,60μmol/L putrescine,30 nmol/L selenium chloride [all from Sigma(USA)except glutamine(sigma)from GIBCO/Invitrogen(USA)] and plated onto T75 flasks(Greiner,Germany)at a density of 1×10(5)cells/ml in the neurosphere medium(basic neurosphere medium supplemented with 20 ng/ml fibroblast growth factor-2)(Invitrogen,USA)and 2μg/ml heparin sulfate(Sigma,USA).Cellswere grown for five to seven days and formed neurospheres.NSC were cultured with 10% fetal bovine serum(FBS)and could differentiate into astrocytes,oligodendrocyte cells,neurons,etc.And we found NSC differentiate into Cholinergic neurons incubated with the extract from feta1 chick leg muscles and TIANTAIⅡ-containing CSF could promote theNSC into cholinergic neurons.
The neurons from the fetal rat(E16-18)hippocampus were culturedwith the medium(includes Neurobasl A medium,2-Mercaptoethanol,EGF,bFGF,Glutamine,penicillin,stepmycin)at 37℃5%CO_2.Thecultured cells were identified as neurons and were used for the followthe experiment.When neurons grew to the logarithmic phase and were incubated with 10μM of Aβ_(25-35)for 24-hour and the AD cell modelwas made And Aβ_(25-35)caused neurons apoptosis and injured MTT method was used for analysis of neurons' growth capacity and AD model.neurons were grown well with TIANTAIⅡ-containing cerebrospinal fluid.With acridine orange-ethidium bromide staining,we counted the value of neirons 1 under fluorescence microscope and calculated the apoptotic index.All results were statistical analyzed.
To sum up,TIANTAI II has a better role in anti-AD of rats,themechanism involves anti-apoptosis,anti-endoplasmic reticulum stress,andpromote the differentiation and transformation of NSC And It can promote the NSC differentiate to cholinergic neurons in particular.Thereis whether or not occurrence of differentially expressed genes,we hopethe further studies on TIANTAIⅡ.
引文
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