【作者】 葛宇松；

【导师】 张朝东；

【作者基本信息】 中国医科大学 ， 神经病学， 2008， 博士

【摘要】 目的阿尔茨海默病（Alzheimer’s disease,AD）是一种常见的中枢神经系统变性疾病,AD临床上表现为不可逆进行性发展的记忆减退、认知、语言障碍及人格的改变等。大量研究发现,AD的主要病理特征是全脑萎缩,脑细胞外出现大量的老年斑（senile plaque,SP）,细胞内神经元纤维缠结（neurofibrillary tangles,NFT）,以及以海马皮层区域为主的神经元大量丢失。β淀粉样蛋白（β-amyloid protein,Aβ）是老年斑的主要成分,且Aβ已经成为AD发病过程中的一个重要因素。体内外实验均显示了Aβ可以直接引起神经细胞的凋亡和死亡,但Aβ诱导神经毒性的机制是复杂的。近年来的研究发现,Aβ可以通过多种不同的途径诱导活性氧产生,产生过多的自由基,引起细胞的氧化应激损伤。已有研究显示,氧化应激反应可以通过一系列的分子机制引起和促进神经元变性并导致神经元丢失,这在Aβ介导的神经毒性过程中起到重要作用。Aβ通过氧化应激反应生成大量的活性氧进一步增加细胞的易损性,从而增加了细胞凋亡的发生。亲环素A（cyclophilin A,CyPA）是亲环素家族成员之一,具有肽脯氨酰顺反异构酶（peptidyl-prolyl cis-trans isomerase,PPIase）活性,它的分子量为18000,也称CyP18。CyPA主要存在于细胞质中,也可出现在细胞核以及细胞外。它是一种高度保守的蛋白质,从低等原核生物到哺乳动物都有这种蛋白,并且广泛分布于许多组织。在脑组织中CyPA高度表达,主要定位于神经元。它参与神经元分化以及成年皮质可塑性,同时过表达CyPA可以增加人胚胎脑细胞的生长率。近年来研究发现,神经细胞以及其他组织细胞遭受氧化应激以及缺血缺氧等损伤时,细胞内的CyPA表达明显升高。此外,细胞内过表达CyPA以及应用外源性的CyPA可以保护神经元抵抗氧化应激以及缺血缺氧等损伤,而降低了细胞内CyPA的表达使细胞更易遭受有害物质的损伤。由此可见,一方面,细胞损伤后CyPA表达升高是一种内源性的保护机制,参与细胞损伤的应答;另一方面,过表达CyPA以及外源性的CypA可以保护细胞,抵抗氧化应激等不良因素造成的损伤,也说明CyPA具有抗氧化的作用。本研究应用Aβ_25-35注入大鼠双侧海马组织建立AD的动物模型以及应用Aβ_25-35干预PC12细胞建立细胞模型并用CypA进行预处理。一方面观察Aβ_25-35注入大鼠海马组织后,所引起的大鼠海马组织的形态和CA1区神经元数量的变化和细胞凋亡情况,并观察海马组织中CypA的mRNA和蛋白表达的变化;另一方面观察CyPA对Aβ_25-35诱导PC12细胞的存活率、细胞形态和凋亡率的影响,以及对线粒体跨膜电位,细胞内ROS水平,细胞的SOD和GSH-Px的活性、凋亡相关基因Bcl-2、Bax和p53及p38MAPK通路的的影响。方法动物实验:取健康的Wistar大鼠60只,随即分为实验组和对照组。每组又按注液后1d,7d和14d分为3个亚组,每组10只。采用双侧海马立体定位注射的方法建立动物模型,实验组每侧海马注射2μl（10μg）Aβ_25-35(Aβ_25-35临用前于37℃放置2d使其凝聚),对照组在海马内注射等量的生理盐水。取各个组其中5只大鼠,于注射后1d,7d和14d相应时间点,进行脑灌流固定,分别进行HE染色,Nissel染色和TUNEL染色。取各个组另外5只大鼠,于注射后1d,7d和14d相应时间点,快速取出大脑,分离双侧海马组织,-70℃冰箱保存待进行CyPA的PT-PCR和Western Blot检测。培养细胞:将培养的PC12细胞,用不同终浓度的CyPA（0.1、1、10和100nmol/L）处理30min,再加入10μmol/L的Aβ_25-35(Aβ_25-35临用前于37℃放置2d使其凝聚)继续培养。每次实验均分为正常对照组(0μmol/L Aβ_25-35)、处理组(10μmol/L Aβ_25-35)和药物保护组(CyPA+Aβ_25-35)。加入Aβ_25-3548小时后,各组采用MTT法检测细胞的存活率,HE染色观察细胞形态,PI单染后流式细胞仪检测凋亡,Hoechst33258荧光染色观察凋亡,RT-PCR和Western Blot的方法检测Bcl-2、Bax和p53基因的mRNA和蛋白的表达。加入Aβ_25-3524小时后,各组采用Rh123荧光染色观察线粒体跨膜电位改变并用流式细胞仪进行定量分析,采用DCF-DA荧光染色观察细胞内ROS的水平并用流式细胞仪进行定量分析,检测细胞内抗氧化酶SOD和GSH-Px的活性,以及Western Blot检测p38MAPK和p-p38MAPK蛋白的表达观察p38MAPK通路的活化情况。结果1、大鼠海马HE染色结果对照组各亚组大鼠海马CA1区细胞带完整,未见明显的神经元缺失,细胞排列有序,未见明显的小胶质细胞增生。实验组1d亚组大鼠海马CA1区较对照组神经细胞略减少,细胞排列略有改变;实验组7d亚组大鼠海马CA1区较对照组神经细胞减少明显,细胞排列紊乱;实验组14d亚组大鼠较7d亚组上述表现更为明显。2、大鼠海马Nissel染色结果对照组各亚组大鼠海马CA1区细胞带无明显受损,神经细胞密集,排列完整。实验组1d亚组大鼠海马CA1区较对照组神经细胞排列不整齐,细胞有所减少;实验组7d亚组大鼠海马CA1区较对照组神经细胞排列紊乱,细胞带结构丧失,细胞减少明显;实验组14d亚组大鼠较7d亚组上述表现更为明显。3、大鼠海马TUNEL染色结果对照组各亚组大鼠海马CA1区几乎未见TUNEL染色阳性细胞。实验组1d亚组凋亡细胞数较少,实验组7d,14d亚组凋亡细胞明显增多,且随时间延长,凋亡数逐渐增多,3个亚组凋亡细胞较对照组均有显著性差异。4、大鼠海马CyPA的mRNA和蛋白的表达情况对照组各亚组大鼠海马均有CyPA的mRNA和蛋白的表达,各组之间没有明显差异。实验组1d亚组的CyPA的mRNA和蛋白的表达明显增高与对照组相比具有显著性差异;实验组7d亚组的CyPAmRNA和蛋白的表达较实验组1d亚组有所下降,但是仍然高于对照组,与对照组相比具有显著性差异;实验组14d亚组的CyPA的mRNA和蛋白的表达进一步下降,且低于对照组,CyPA的mRNA的表达较对照组没有显著差异,但是蛋白的表达较对照组有显著差异。5、MTT检测细胞存活率Aβ_25-35处理组细胞存活率较正常对照组明显降低,而药物保护组与Aβ_25-35处理组相比能明显提高的细胞存活率,除0.1nmol/L的CyPA的作用不明显,其余各组细胞存活率均明显提高,具有浓度依赖性。6、细胞的HE染色结果正常PC12细胞胞体饱满,细胞间联系紧密,胞核蓝紫色,Aβ_25-35处理组的细胞数量减少,胞浆皱缩或饱涨,胞质浓缩,核固缩或碎裂,呈蓝黑色,10和100nmol/L的CyPA预处理组的大多数细胞形态接近正常。7、PI单染后流式细胞仪检测凋亡的结果正常对照组有少量的细胞凋亡,Aβ_25-35处理组细胞凋亡率明显增加,药物保护组中1、10和100nmol/L的CyPA明显降低了细胞的凋亡率,而0.1nmoI/L的CyPA几乎不起作用。8、Hoechst33258染色结果正常对照组细胞核呈现弥散均匀的蓝色荧光;Aβ_25-35处理组可见明显的细胞凋亡,呈现高亮蓝色浓染致密的颗粒块状荧光,细胞核明显固缩、凝聚和断裂;除了0.1nmol/L的CyPA组以外,其余各CyPA预处理组的凋亡的细胞明显减少。9、线粒体跨膜电位的检测结果Aβ_25-35处理组细胞线粒体跨膜电位较正常对照组明显降低,表现为细胞的荧光强度降低,1、10和100nmol/L的CyPA处理组较Aβ_25-35处理组明显提高了细胞线粒体跨膜电位,而0.1 nmol/L的CyPA作用不显著。10、细胞内活性氧的检测结果Aβ_25-35处理组细胞内ROS水平较正常对照组明显升高,表现为细胞的荧光强度增强,1、10和100nmol/L的CyPA处理组明显抑制了Aβ_25-35诱导的细胞内ROS的增多,而0.1 nmol/L的CyPA不起作用。11、SOD和GSH-Px活性的检测结果Aβ_25-35处理组细胞内SOD和GSH-Px活性较正常对照组明显降低,10和100nmol/L的CyPA处理组明显提高了细胞SOD和GSH-Px活性,较Aβ_25-35处理组有显著性差异,1 nmol/L的CyPA处理组SOD活性较Aβ_25-35处理组没有显著性差异,但是GSH-Px活性较Aβ_25-35处理组有显著性差异,而0.1 nmol/L的CyPA几乎不起作用。12、Bcl-2和Bax基因mRNA和蛋白表达结果Aβ_25-35处理组Bcl-2基因的mRNA和蛋白表达较正常对照组明显减少,Bax基因的mRNA和蛋白表达明显增加。除0.1nmol/L的CyPA处理组外,其余各CyPA处理组Bcl-2基因的mRNA和蛋白表达较Aβ_25-35处理组明显增加,Bax基因的mRNA和蛋白表达较Aβ_25-35处理组明显减少,呈剂量依赖性。Aβ_25-35处理组Bcl-2和Bax基因mRNA和蛋白比值较正常对照组明显减低,CyPA处理后,除0.1nmol/L的CyPA处理组外,其余各组的比值均明显增高,呈剂量依赖性。13、p53基因的的mRNA和蛋白表达结果Aβ_25-35处理组p53基因的mRNA和蛋白表达明显增加。除0.1nmol/L的CyPA处理组外,其余各CyPA处理组p53基因的mRNA和蛋白表达较Aβ_25-35处理组明显减少,呈剂量依赖性。14、p38MAPK通路活化情况正常对照组未见p38MAPK通路活化,Aβ_25-35处理后p38MAPK通路明显活化,1、10和100nmol/L的CyPA可以抑制Aβ_25-35诱导的p38MAPK通路活化,而0.1 nmol/L的CyPA未见明显作用。结论1、在大鼠海马部位立体定位注射Aβ_25-35后,引起海马神经元受损并且导致凋亡,随时间延长,损伤增强。2、在大鼠海马部位立体定位注射Aβ_25-35后,引起海马组织的CyPAmRNA和CyPA蛋白水平表达改变,早期增加,但随时间延长,表达逐渐减少。3、CypA可以通过抵抗诱导的PC12细胞线粒体跨膜电位的降低,ROS生成增加和SOD和GSH-Px的活性降低保护细胞,提高PC12细胞存活率,降低凋亡率。4、CypA可以通过抵抗Aβ_25-35诱导的PC12细胞Bcl-2mRNA和蛋白减低,Bax和p53的mRNA和蛋白减低以及抑制p38MAPK通路的活化参与细胞保护,减少凋亡。更多还原

【Abstract】 ObjectiveAlzheimer’s disease（AD） is a common central neurodegenerative disease.The clinical manifestation of AD include inreversible progressive failure of memory, disorder of recongnition and language and change of personality.A lot of study show that the pathological feature of AD is atrophy of brain,senile plaque（SP） appeared out of cells,neurofibrillary tangles and loss of neurons at hippocampus and cortex.β-amyloid protein（Aβ） is the main component of SP,and it has been an important factor of genesis of AD.Experiments in vivo and in vitro demonstrate Aβcan induce apoptosis and death of neurons,but the mechanism of Aβinduced-neurotoxity is complex.Recently,study has shown Aβcould incuce overproduction of free radical by different pathway and cause oxidative stress induced-injury in cells.Several studies suggest that the oxidative stress play a key role in Aβ-mediated neuronal cytotoxicity by triggering or facilitating neurodegeneration through a wide range of molecular events which eventually lead to neuronal cell loss.Aβ-induced reactive oxidative species（ROS） overproduction can increas the vulnerability of cells and in turn increase apoptotic cell death.cyclophilin A（CyPA） is a member of cyclophilin family,which has the activity of peptidyl-prolyl cis-trans isomerase.The molecular weight of CyPA is 18000,so it is also named CyP18.CyPA occurs cytosolically,within the nucleus and extracellularly.It is a highly conservative protein,appears in prokaryotic organism and mammalian.It is widely distributed in many tissues.In the brain,CyPA displays the high expression, where it is predominantly localized to neurons.CyPA is implicated in neuronal differentiation and adult cortical plasticity.Moreover,CyPA over-expression increases growth rates of human embryonic brain cells.Recently,study showed that increase in expression of CyPA was occurred in neural cells and cells from other tissues which were suffered oxidative stress,ischemic or hypoxemic injury.Moreover, overexpression of CyPA and exogenic CyPA can protect neurons agaist oxidative stressinjury and ischemic and hypoxemic injury.But CyPA down-regulation renders cells more susceptible to the injury of noxious substance.In one hand,it is a endogenicly protective mechnism that CyPA is up-regulated after cells injury as response to cells injury.In the other hand,overexpression of CyPA and exogenic CyPA can protect cells agaist oxidative stress-induced injury.That is to say,CyPA has the antioxidative effect.In our expriments,we set up a rat AD model by injection Aβinto bilateral hippocampus of rat and a cell model through using PC12 cells incubated by Aβwhich is pretreated by CyPA.In one hand,we observe the morphological feature of hippocampus,the number of neuron and apoptotic cells in CA1 area and the change of CypAmRNA and protein in hippocampus are detected after Aβ_25-35 was injected.In the other hand,we observe the effect of CyPA on Aβ_25-35-induced survival rate, morphological feature of cells and apoptotic rate and transmembrane potential,the lever of cellular ROS,activity of SOD and GSH-Px,apoptosis relative gene Bcl-2,Bax and p53 as well as p38MAPK pathway are studied.MethodsAnimal experiment:60 healthy Wistar rats were divided into experimental group and control group randomly.In each group there were three subgroup -1d subgroup,7d sungroup and 14d subgroup-according to the time after injection of Aβ_25-35.In every sungroup there were 10 rats.The model was established through injection into bilateral hippocampus,injection of 2μl（10μg） Aβ_25-35(Aβ_25-35 was aggregated at 37℃for 2 days) in the experimental group and injection of NS in the control group.The brains of 5 rats in every group were perfused and fixed at 1d,7d and 14d after injection,and the samples of brain were stained with HE,Nissel and TUNEL.The other 5 rats were killed at 1d,7d and 14d after injection.The brains were taken quickly,the hippocampi were separated and preserved in refrigerator at -70℃,then were detected the expression of CyPA by RT-PCR and Western Blot.Cell culture:The PC12 cells were pretreated with CyPA（0.1,1,10 and 100nmol/L） for 30 minutes,then incubated with Aβ_25-35(Aβ_25-35 was aggregated at 37℃for 2 days). In every experiment there were normal control group(0μmol/L Aβ_25-35),treatment group(10μmol/L Aβ_25-35) and drug-protective group(CyPA+Aβ_25-35).After incubation with Aβ_25-35 for 48 hours,the PC12 cells were detected the survival rate by MTT method,observed the morphological feature by HE stain and apoptosis by PI stain and Hoechst 33258 stain,detected the expression of mRNA and protein of Bcl-2,Bax as well as p53 by RT-PCR and Western Blot.After incubation with Aβ_25-35 for 24 hours, the PC12 cells were detected transmembrane potential by Rh123 stain,evaluated the level of ROS in cells by DCF-DA stain as well as the activities of SOD and GSH-Px, detected the activation of p38MAPK pathway by observation the protein’s expression of p38MAPK and p-p38MAPK through Western Blot.Results1.HE stain of rat hippocampusIn control group,the cell zone was complete in CA1 area of rat hippocampus.The loss of neurons and the proliferation of microglia cells were not seen and the arrangement of cells were regular.In 1d subgroup of experimental group,the number of cells was decreased compared with control group and the arrangement of cells appeared alteration slightly.In 7d subgroup of experimental group,the decrease of cells was more obviois and the cells were arranged unregular.In 14d subgroup of experimental group,the above-mentioned changes were more significant than that in 7d subgroup.2.Nissel stain of rat hippocampus In control group,the cell zone was not injured in CA1 area of rat hippocampus. We could see the close neurons and complete arrangement.In 1d subgroup of experimental group,arrangement of the cells was tmregular slightly,the decreas of cells appeared.In 7d subgroup of experimental group,the arrangement of cells was disordered,the contruction of cell zone was disappeared and the number of cells was diminished.In 14d subgroup of experimental group,the above-mentioned changes were more significant than that in 7d subgroup.3.TUNEL stain of rat hippocampusIn the control group,we saw few TUNEL-positive cells.In 1d subgroup of experimental group,there was several TUNEL-positive cells.In 7d and 14d subgroup of experimental group,there were many apoptotic cells.With the prolong of time,the number of apoptotic cells was increased.We could see the significant difference between experimental group and control group.4.The expression of CyPAmRNA and proteinThe expression of CyPAmRNA and protein were appeared in every subgroup of control group,and there was no significant difference among 3 subgroups.In 1d subgroup of experimental group,the expression of CyPAmRNA and protein were increased dramaticly and there was significant difference compared with control group. In 7d subgroup of experimental group,the expression of CyPAmRNA and protein were lower than that in 1d subgroup,but they were higher than that in control group and there was a significant difference compared with control group.In 14d subgroup of experimental group,the expression of CyPAmRNA and protein decreased,and they were lower than control group.There was no significant difference compared the expression of CyPAmRNA with that in control group,but there was a significant difference compared the expression of CyPA protein with that in control group.5.Cell viability detected by MTT methodThe cell viability in Aβ_25-35 treatment group was lower than that in control group. The cell viability in drug-protective group was increased compared with Aβ_25-35 treatment group.Except o.1nmol/L CyPA,other CyPA with concentration of lnmol/L-100nmol/L could rise the cell viability significantly and it was concentration-dependent.6.HE stain of cellsWe could see full cell body,tight intercellular junction and blue-purple neucli in cultured PC12 cells.In Aβ_25-35 treatment group,decrease in the number of cells, shrinkage or swell of cytoplasm,condense of cytoplasma,karyopyknosis,karyorrhexis and blue-black neucli could been seen.Pretreatment with 10nmol/L and 100nmol/L CyPA could protect PC12 cells and make the morphological feature get close to normal.7.Apoptosis was detected by flow cytometry with PI stainThere were slight apoptotic cells in normal control group.In Aβ_25-35 treatment group,the apoptotic cells were increased obviously,which could been diminished by treatment with 1,10 and 100nmol/L CyPA.But 0.1 nmol/L CyPA could not prevent the increase of apoptotic cells induced by Aβ_25-35.8.Hoechst33258 stainNormal PC12 cells appeared diffusedly and uniformly blue flourescence.In Aβ_25-35 treatment group,there were significant apoptotic cells appearing sapphirine and condensed granular flourescence and the neucli appeared condensed,aggregated and disrupted.Except 0.1nmol/L CyPA,other CyPA with concentration of 1,10 and 100nmol/L could decrease apoptosis of cells.9.Transmembrane potentialThe transmembrane potential in Aβ_25-35 treatment group was decreased compared with control group,which manifestated that the intensity of flourescence decreased. CyPA with concentration of 1,10 and 100nmol/L could increased the transmembrane potential,but 0.1 nmol/L CyPA play no role. 10.Intracellular ROSThe level of ROS in Aβ_25-35 treatment group was increased compared with control group.CyPA with concentration of 1,10 and 100nmol/L could inhibit the increase of ROS induced by Aβ_25-35.There was no significant change in 0.1nmol/L CyPA treatment group.11.Activities of SOD and GSH-PxActivities of SOD and GSH-Px were decreased by treatment with Aβ_25-35.CyPA with concentration of 10 and 100nmol/L could increase the activities of SOD and GSH-Px and there was a significant difference compared with Aβ_25-35 treatment group. We could see it is no statistical significance that,activity of SOD in 1nmol/L CyPA treatment group compared with that in Aβ_25-35 treatment group,but there was a siginificant difference in the activity of GSH-Px.0.1nmol/L CyPA play no role.12.The expression of mRNA and protein about Bcl-2 and BaxWhen the cells were incubated with Aβ_25-35,the expression of mRNA and protein of Bcl-2 decreased and the expression of mRNA and protein of Bax increased.Except 0.1 nmol/L CyPA,other CyPA with concentration of 1,10 and 100nmol/L could inhibit Aβ_25-35-induced the decrease of Bcl-2 and the increase of Bax concentration-dependently.The ratio of Bcl-2 and Bax in mRNA and protein was reduced when the PC12 cells were incubated with Aβ_25-35.Except 0.1nmol/L CyPA,other CyPA with concentration of 1,10 and 100nmol/L could prevent Aβ_25-35-induced the decrease of the ratio concentration-dependently.13.The expression of mRNA and protein about p53In Aβ_25-35 treatment group,the expression of p53mRNA and protein increased which could been inhibited by CyPA with concentration of 1,10 and 100nmol/L concentration-dependently,but could not been prevented by 0.1nmol/L CyPA.14.The activation of p38MAPK pathway There was no activation of p38MAPK pathway in control group.Aβ_25-35 could activat p38MAPK pathway,which could been inhibited by CyPA with concentration of 1,10 and 100nmol/L,not 0.1 nmol/L CyPA.Conclusions1.After injection Aβ_25-35 into hippocampus of rat,the neurons in hippocampus of rat were injuried and occurred apoptosis.With the prolong of time,the injury enhanced.2.After injection Aβ_25-35 into hippocampus of rat,the expression of CyPAmRNA and protein were changed.In the early stage the expression increase and in turn,the expression decreased gradually.3.CyPA protect PC12 cells agaist Aβ_25-35-induced decrease of viability and increase of apoptotic rate through increase of the transmembrane potential,decrease of ROS and increase of activities of SOD and GSH-Px.4.CyPA protect PC12 cells agaist Aβ_25-35-induced apoptosis through increase of the expression of Bcl-2,decrease of the expression of Bax and p53 and preventation of activation of p38MAPK pathway.更多还原