【作者】 吴小妹；

【导师】 肖波； 黄志凌；

【作者基本信息】 中南大学 ， 神经病学， 2009， 博士

【摘要】 第一部分微管蛋白在颞叶癫癇模型突触后致密物中的动态表达目的:探讨微管蛋白α-tubulin与β-tubulin在颞叶癫癇大鼠模型突触后致密物(PSD)中的动态表达。方法:健康成年雄性Sprague-Dawley(SD)大鼠,体重250±20 g,200只,实验随机分为对照组和颞叶癫癇(TLE)组。建立氯化锂-匹罗卡品颞叶癫癇大鼠模型,分别在癫癇持续状态(SE)后1d、3d、7d、15d、30d 6个时间点提取全脑组织,分离PSD蛋白。应用westernblot方法检测β-actin、β-tubulin及GAPDH蛋白在PSD中的表达变化,确定PSD蛋白western blot半定量分析的内参;采用western blot及免疫电镜方法检测微管蛋白α-tubulin与β-tubulin在PSD中的表达变化。结果:1.氯化锂-匹罗卡品大鼠致癇模型:SE诱发成功率为91.1%,总死亡率为20.6%,模型成功率为70.6%。2.PSD蛋白纯度:NR1与PSD-95蛋白在P2成分、突触小体及PSD中均呈免疫印迹阳性,而SynPhy蛋白在PSD成分中呈阴性。3.颞叶癫癇大鼠PSD蛋白western blot半定量分析的内参确定:β-actin、β-tubulin及GAPDH在对照组中均表达,β-actin及β-tubulin蛋白含量从SE后1d开始减少,SE后7d降至最低,SE后15d、30d表达有所增多,但仍低于对照组(P<0.01)。而GAPDH蛋白在SE各组及对照组中的表达无明显差异(P>0.05)。随后,我们随机选取正常对照组及SE后15天PSD蛋白两个样本,每个样本置6个泳道,分别对这三个蛋白进行分析。实验证实与正常对照组的6个泳道蛋白相比,SE后15天6个泳道的蛋白β-actin与β-tubulin的表达均明显减少,有明显的统计学意义(P<0.01)。而GAPDH的表达在所有泳道中的表达则无明显差异(P>0.05),提示β-actin与β-tubulin的表达变化与加量误差无关。4.微管蛋白α-tubulin与β-tubulin在PSD的表达检测:①α-tubulin与β-tubulin在SE后1d、3d、7d、15d、30d的表达明显减少,SE后7d降至最低(P<0.01),在SE后15d及30d蛋白含量较SE后7d时有所增多,但仍明显低于对照组(P<0.01)。②免疫电镜结果显示在正常对照组海马CA1中,β-tubulin蛋白在突触前、突触问隙、突触后及胞浆中均有表达。与对照组比较,PSD中β-tubulin在SE后1d、3d、7d、15d、30d PSD中的阳性表达颗粒明显减少,SE后7d降至最低(P<0.01),几乎未见有阳性颗粒表达,SE后15d及30d表达颗粒有所增多,当仍明显低于对照组(P<0.01)。结论:1.GAPDH蛋白更适合做颞叶癫癇大鼠PSD蛋白westernblot半定量研究的内参。2.PSD蛋白tubulin参与了颞叶癫癇形成机制。第二部分突触后致密物微管蛋白对颞叶癫癇大鼠自发发作的影响目的:探讨突触后致密物微管蛋白α-tubulin与β-tubulin对颞叶癫癇大鼠反复自发发作形成的影响。方法:健康成年雄性Sprague-Dawley(SD)大鼠,体重250±20g,236只,实验随机分为对照组、TLE自发发作(TLE-SRS)组和TLE无自发发作(TLE-NSRS)组。建立氯化锂-匹罗卡品颞叶癫癇大鼠模型,提取全脑组织,分离PSD蛋白。采用western blot及免疫电镜方法检测微管蛋白α-tubulin与β-tubulin在PSD中的表达变化。结果:1.氯化锂-匹罗卡品致癇模型SE诱发成功率为91%,总死亡率为22%,模型成功率为72.5%,自发发作率为50.5%。TLE-SRS组大鼠EEG异常,表现θ节律增多及单个、多个棘波或阵发连续棘波群发放;TLE-NSRS组大鼠EEG表现为正常或轻度异常,有时可发现散在的棘波或尖波;生理盐水对照组EEG亦未见癫癇样放电波。2.尼氏染色:与对照组比较,TLE-SRS组海马CA1、CA3区及齿状回区均有显著的神经元减少(P<0.01)。CA1区和CA3区锥体细胞层神经元排列紊乱,可见部分神经元形态不完整,细胞肿胀或皱缩、轮廓模糊、界限不清,细胞间距加大,胞浆尼氏小体减少。齿状回颗粒细胞边界不整齐、排列松散、增宽,呈现颗粒细胞发散改变。TLE-NSRS组存活神经元与对照组相比也有明显减少,但较SRS组要多(P<0.01)。3.微管蛋白α-tubulin与β-tubulin在PSD的表达检测:①与对照组比较,TLE-NSRS组α-tubulin在PSD中表达明显减少(p<0.01),TLE-SRS组PSD中α-tubulin含量较对照组也显著减少(p<0.01);与NSRS组相比,其表达水平也明显降低(p<0.01)。②与对照组比较,TLE-NSRS组及SRS组β-tubulin在PSD中表达明显减少(p<0.01);与NSRS组相比,SRS组PSD中β-tubulin含量也明显降低(p<0.01)。免疫电镜结果显示在正常对照组海马CA1中,β-tubulin蛋白在突触前、突触间隙、突触后及胞浆中均有表达,我们将着重与突触后致密物PSD中β-tubulin在SE后的表达变化。与对照组比较,蛋白β-tubulin在TLE-NSRS组及SRS组PSD中的阳性表达颗粒明显减少(p<0.01),与NSRS组相比,SRS组PSD中β-tubulin含量也明显降低(p<0.01)。支持western blot分析结果。结论:PSD中Tubulin蛋白表达与颞叶癫癇反复自发发作呈负相关。第三部分海马微管稳定性对颞叶癫癇成的影响目的:探讨干预微管的稳定性对颞叶癫癇形成的影响。方法:①健康成年雄性SD大鼠,体重250±20g,75只,立体定向海马注射不同剂量的秋水仙碱(微管解聚剂)及紫杉醇(微管聚合剂),根据尼氏染色、免疫组织化学检测β-tubulin的表达确定秋水仙碱及紫杉醇的最佳注射剂量。②健康成年雄性SD大鼠,体重250±20g,60只,建立氯化锂-匹罗卡品颞叶癫癇大鼠模型,在SE后7天,分别予以立体定向海马注射最适剂量的秋水仙碱及紫杉醇,归于秋水仙碱干预组及紫杉醇干预组。对各组大鼠进行行为学观察及脑电图记录,30天后根据有无自发发作每组又分为自发发作组及无自发发作组两亚组。采用免疫电镜方法检查β-tubulin在PSD中的表达。结果:1.海马注射秋水仙碱最适剂量的确定:①尼氏染色:与对照组相比,秋水仙碱0.01、0.03、0.1、0.3ug干预组存活神经元数量无明显变化(p>0.05),1.0与2.0ug治疗组神经元死亡明显增多(p<0.01),细胞排列紊乱,可见神经元形态不完整,细胞肿胀或皱缩、轮廓模糊、界限不清。②免疫组织化学方法检测发现对照组可见β-tubulin大量表达,主要分布在胞浆,与对照组比较,秋水仙碱0.01、0.03、0.1ug治疗组β-tubulin表达无明显差异(p>0.05),0.3ug治疗组表达减少(p<O.01),1.0与2.0ug治疗组明显减少(p<0.01)。③提示海马注射秋水仙碱的最佳剂量为0.3ug,使微管解聚,但不影响神经元细胞的存活。2.海马注射紫杉醇最适剂量的确定:①尼氏染色:与对照组相比,紫杉醇0.1、0.5、1.0ug干预组存活神经元数量无明显变化(p>0.05),1.5ug治疗组神经元死亡明显增多(p<0.01),细胞排列紊乱,可见神经元形态不完整,细胞肿胀或皱缩、轮廓模糊、界限不清。②免疫组织化学方法检测发现与对照组比较,紫杉醇0.1、0.5ug治疗组β-tubulin表达无明显差异(p>0.05),1.0ug治疗组表达较对照组增多(p<0.05),但1.5ug治疗组表达明显减少(p<0.01)。③提示海马注射紫杉醇的最佳剂量为1.0ug,使微管聚合,但不影响神经元细胞的死亡。3.秋水仙碱0.3ug干预组及紫杉醇1.0ug干预组大鼠行为学及EEG与对照组无差异,提示秋水仙碱及紫杉醇无致癇性。4.TLE组白发发作率为75%,秋水仙碱组自发发作率为90%,。紫杉醇组自发发作率为40%。脑电图结果显示TLE-SRS组大鼠EEG重度异常,表现θ节律增多及单个、多个棘波或阵发连续棘波群发放,TLE-NSRS组大鼠EEG正常或轻度异常,偶见单个棘波或尖波发放,与TLE-SRS组比较,秋水仙碱-SRS组大鼠EEG无显著差异,而紫杉醇-SRS组大鼠EEG棘尖波明显减少,频率及波幅均降低。秋水仙碱-NSRS组及紫杉醇-NSRS组大鼠EEG与TLE-NSRS组相比无显著差异。5.微管蛋白β-tubulin在PSD的表达检测:免疫电镜结果显示与TLE-SRS组比较,秋水仙碱-SRS组β-tubulin表达无显著差异(p>0.05),而紫杉醇-SRS组表达明显增多(p<0.01)。秋水仙碱-NSRS组与紫杉醇-NSRS组与TLE-NSRS组比较β-tubulin表达无显著差异(p>0.05)。结论:1.海马微管稳定性障碍是颞叶癫癇形成的易感因素;2.促微管聚合剂可能可防止颞叶癫癇形成。更多还原

【Abstract】 Part One The down-regulation of microtubules at postsynaptic density in rats with temporal lobe epilepsyObjective To investigate the expression change of proteinsα-tubulin andβ-tubulin at postsynaptic density(PSD) in rats with temporal lobe epilepsy(TLE).Method 200 male Sprague-Dawley(SD) rats were randomly divided into control group and TLE group.The lithium-pilocarpine model of TLE was established.,The whole brain was collected to isolate the PSD at 1d,3d,7d,15d and 30d after SE.First,the expression of proteinsβ-actin、β-tubulin and GAPDH were detected using Western Blot to define the internal control of Western Blot analysis of proteins at PSD. Then,Western Blot and immuno-electron microscope analysis were used to detect the expression of microtubule proteinsα-tubulin andβ-tubulin at PSD.Results 1.The lithium-pilocarpine model of TLE:SE was induced at the achievement ratio of 91.1%and total mortality of 20.6%.2.Purity of PSD:The proteins NR1 and PSD-95 were all expressed in P2 fraction,synaptosome and PSD fraction,but Synphy protein was absent in PSD fraction.3.Definition of internal control in western blot analysis of rat PSD with TLE:The proteinsβ-actin,β-tubulin and GAPDH were all expressed in control group.β-actin andβ-tubulin were decreased from 1d after SE, and were slowest on 7d after SE(P＜0.01).Compared to on 7d after SE, the two proteins were increased on 15d and 30d after SE,but still less than control group(P＜0.01).The expression of GAPDH in TLE group was no significant difference compared to control group(P＞0.05).After then, we randomly chose two PSD samples from control group and 15-day SE group.Each sample were loaded on six lanes.Our results showed that the expression ofβ-actin andβ-tubulin were significantly lower in six lanes of the SE group than in the control group(P＜0.01).Whereas the expression of GAPDH had no difference in two samples(P＞0.05).It was indicated that the variation ofβ-actin andβ-tubulin expression was a result of TLE instead of loading error.4.The expression ofα-tubulin andβ-tubulin at PSD①the expression ofα-tubulin andβ-tubulin were markly decreased from the time point of 1 day after SE,and were lowest on 7-day after SE(P＜0.01). The protein levels on 15-day and 30-day after SE were higher than on 7-day after SE,but still largely lower than in control group(P＜0.01).②Immuno-electron microscopy showed that in CA1 part of hippocampus in control group,β-tubulin was expressed in cytoplasm and synapse including presynapse,synaptic cleft and PSD.Our study emphasized on the change of expression ofβ-tubulin at PSD.Immunogold labeled particles ofβ-tubulin were markly decreased from 3-day after SE,and decreased lowest on 7-day after SE(P＜0.01).And then the expression was increased from 15-day after SE compared to on 7-day after SE,but still less than in control group(P＜0.01).Conclusion 1.GAPDH was more suitable for the internal control in western blot analysis of rat PSD with TLE.2.Microtubulesα-tubulin andβ-tubulin may be one of the mechanism in the formation of temporal lobe epilepsy. Part Two Effect of microtubules at postsynaptic density on spontaneous seizure of rat with temporal lobe epilepsyObjective To investigate the effect of microtubulesα-tubulin andβ-tubulin at postsynaptic density(PSD) on recurrent spontaneous seizure of rat with temporal lobe epilepsy(TLE).Method 236 male SD rats were randomly used to set up lithium-pilocarpine model of TLE and decided into control group,TLE with recurrent spontaneous seizure(SRS) group and TLE with no recurrent spontaneous seizure(NSRS) group.The whole brain was removes to isolate the PSD.The expression ofα-tubulin andβ-tubulin was detected with western blot and immuno-eletron microscopic analysis.Results 1.The lithium-pilocarpine model of TLE SE was induced at the achievement ratio of 91.1%,total mortality of 20.6%and spontaneous seizure ration of 50.5%.EEGs of rats in TLE-SRS group were abnormal,presenting moreθwave,single or multiple spikes and groups of spikes.EEGs of rats in TLE-NSRS group were normal or mild abnormal,with sporadic spikes and sharp waves.2.Niss1 staining Compared with the control group,the numbers of surviving neurons in regions of CA1,CA3 and dentate gyms were markedly decreased in TLE-SRS group(p＜0.01).We found that the neurons with disorder,cell swelling or shrinkage,irregular border and decrease of Nissl bodies in cytoplasm.The surviving neurons in TLE-NSRS group were markly decreased compared to control group (p＜0.01),but more than in TLE-SRS group(p＜0.01).3.The expressions ofα-tubulin andβ-tubulin at PSD①Compared to control group,the expressions ofα-tubulin andβ-tubulin at PSD in TLE-SRS group and TLE-NSRS group were largely decreased(p＜0.01). And the expressions of these proteins in TLE-SRS group were also lower than in TLE-NSRS group(p＜0.01).②Immuno-eletron microscopy analysis The immunogold labeled particles ofβ-tubulin at PSD were significantly decreased in TLE-SRS and TLE-NSRS group compared to in control group(p＜0.01).And the particles in TLE-SRS group were also less than in TLE-NSRS group(p＜0.01).Conclusion expression of microtubules may be negatively related to recurrent spontaneous seizures of temporal lobe epilepsy. Part Three Effect of microtubule stability on temporal lobe epilepsyObjective To observe the effect of intervention in microtubule stability on temporal lobe epilepsy.Method①75 male SD rats were randomly used to make stereotactic infusion of hippocampus with different doses of cochicine and paclitaxol respectively and divided into control group, cochicine-treated group and paclitaxel-treated group.Nissl staining and detection ofβ-tubulin with immnohistochemistry were made to define the suitable infusion dose of the two drug.②60 male SD rats were randomly set up lithium-pilocarpine model of TLE.At the time point of 7-day after SE,the suitable dose of cochincine and paclitaxel were stereostacticly infused hippocampally respectively,which were divided into TLE-control group,TLE-cochincine group and TLE-paclitaxel group.We observed rats in three group behaviourally for 30 days,and 30-day after SE we made EEG of rats.Then each group was divided into spontaneous recurrent seizure(SRS)group and no spontaneous recurrent seizure (NSRS)group again according to whether or not the rats had recurrent spontaneous seizures.Immuno-electron microscopy was used to detect the expression ofβ-tubulin.Results 1.The definition of suitable infusion dose of cochicine①Nissl staining:compared to control group,the surviving neurons were no significant difference in cochicine-treated group with the infusion dose of 0.01,0.03,0.1,0.3ug(p＞0.05),and were markly decreased with the dose of 1.0 and 2.0ug(p＜0.01).②Immunohistochemistry:β-tubulin was largely expressed in cytoplasm in control group.The expression ofβ-tubulin was no difference in cochicine-treated group with the dose of 0.01,0.03,0.1ug compared to control group and was markly decreased from the dose of 0.3ug(p＜0.01).③Our results indicated that the suitable infusion dose of cochicine was 0.3ug,which induced microtubule depolymerization but did not affect the survival of neurons.2.The definition of suitable infusion dose of paclitaxol①Nissl staining:compared to control group,the surviving neurons were no significant difference in paclitaxel-treated group with the infusion dose of 0.1,0.5,1.0ug(p＞0.05),and were markly decreased with the dose of 1.5ug(p＜0.01).②Immunohistochemistry:Compared to control group,The expression ofβ-tubulin was no difference in paclitaxel-treated group with the dose of 0.1,0.5ug and was increased in the dose of 1.0ug(p＜0.01),but markly decreased in the dose of 1.5ug(p＜0.01).③Our results indicated that the suitable infusion dose of paclitaxel was 1.0ug,which induced microtubule polymerization but did not affect the survival of neurons.3.In cochicine 0.3ug -treated group and paclitaxel 1.0ug-treated group,the behavior and EEG recording of rats were no difference compared to control group,indicating that these drugs had no epileptogenesis.4.The recurrent spontaneous seizures ratio of TLE-control group was 75%,TLE-cochicine group 90%and TLE-paclitaxel group 40%. EEGs of rats in TLE-SRS group were abnormal,presenting moreθwave,single or multiple spikes and groups of spikes.EEGs of rats in TLE-NSRS group were normal or mild abnormal,with sporadic spikes and sharp waves.Compares to TLE-SRS group,EEG of rats in TLE-cochicine SRS group were no difference and EEG in TLE-paclitaxel SRS group had less spike and sharp waves,of which frequency and amplitude were decreased.There were no significant difference in TLE-cochicine NSRS group or TLE-paclitaxel NSRS group compared to TLE-NSRS group. 5.The expression ofβ-tubulin at PSD Compared to TLE-SRS group,the immunogold labeled particles ofβ-tubulin at PSD in TLE-cochicine SRS group were no difference(p＞0.05) and particles in TLE-paclitaxel SRS group largely increased(p＜0.01).Compared to TLE-NSRS group,the expression ofβ-tubulin in TLE-cochicine NSRS and TLE-paclitaxel NSRS group was no significant difference(p＞0.05).Conclusion 1.Abnormality of microtubule stability is the predisposing factor of temporal lobe epilepsy.2.microtubule polymerized agent may prevent temporal lobe epilepsy.更多还原