【作者】 郭凤；

【导师】 蔡际群；

【作者基本信息】 中国医科大学 ， 药理学， 2009， 博士

【摘要】 前言电压门控性钠通道为一种跨膜的糖蛋白,其在神经元动作电位的产生与传播过程中发挥重要作用。在中枢神经系统中,钠通道通常由一个α型大亚基（260KD）和一个或多个β型小亚基（32～37KD）组成,其结构均由四个有50%序列同源的结构域（Ⅰ～Ⅳ）组成,每个结构域含有6个跨膜片段（S₁～S₆）。研究表明α型大亚基与β型小亚基均具有对钠通道失活与激活动力学的调节作用;此外β型小亚基可以作为细胞黏附因子来发挥调节作用。哺乳动物的α型大亚基根据不同的组织特异性与生物物理学特性由9个基因编码。中枢神经系统中,Nav1.1、Nav1.2、Nav1.3和Nav1.6表达较多,其中Nav1.1在海马、小脑、脊髓、脑干等部位表达很高。已有钠通道的200多种基因突变在不同癫痫表型的研究中被证实。伴热惊厥性全身性癫痫（Generalized epilepsy withfebrile seizure plus,GEFS⁺）是一种常染色体显性遗传的特发性全身性癫痫,SCN1A（编码Nav1.1的基因）和SCN1B（编码β₁的基因）的突变可引起GEFS⁺。Nav1.3的mRNA在癫痫患者的海马CA4区表达显著升高;而对于癫痫持续状态诱发后4小时的新生鼠,Nav1.3的mRNA在其海马CA1、CA3区以及齿状回颗粒细胞区表达上调。因此,以往很多研究均表明电压门控性钠通道的遗传异常表达可能会引起中枢神经系统神经元的兴奋性变化。自发性癫痫大鼠（Spontaneously epileptic rat,SER）是一种纯合型的双基因突变大鼠:其突变基因tm来自于东京Wistar大鼠突变系Tremor,突变基因zi来自于德国斯普拉格——道利鼠突变系Zitter,两种突变基因均为常染色体隐性遗传。出生后8周,SER自发性地出现癫痫大发作和失神样的小发作,其脑电图典型表现为皮质与海马区出现的5～7赫兹的棘慢复合波。到目前为止,SER被认为是研究人类癫痫最好的动物模型之一,抗癫痫药物对SER的治疗作用反应与对人类癫痫作用十分相近。有报道利用SER进行有关改善天冬氨酸酰酶的基因治疗;而涉及离子通道病方面,仅报道了SER电压门控性钙通道的功能变化。然而,迄今为止,有关SER电压门控钠通道的研究仍为空白。在本研究中,我们假设SER在其遗传性癫痫发生发展中涉及到钠通道的参与与变化。因此,我们拟从基因与蛋白两个层面研究SER电压门控性钠通道的表达情况,以期明确其在癫痫海马兴奋性中的角色与作用。实验材料和方法1、材料动物、试剂与仪器:健康SER和正常Wistar大鼠9～12周龄,每个实验组均为6只;Wistar新生鼠5只,均小于一周龄;雌雄不限,均由中国医科大学实验动物部提供。Trizol（GIBCO BRL）,免疫组化SP试剂盒（北京中山公司）,实时定量RT-PCR试剂盒（Takara）,引物由上海生工生物工程公司合成,其他药物均为国产分析纯。Ⅰ型与Ⅲ型钠通道亚基兔多克隆抗体（anti-Nav1.1,anti-Nav1.3,Sigma）,anti-β_1ex由密歇根大学Lori L.Isom博士赠送;CM1800 LEICA冰冻切片机（德国）,PCR仪（PE-9600,PE-2400）,凝胶自动成像仪（美国,GDSH型）,MetaMor2ph/DP10/BX41图像分析系统（美国）。2、方法RT-PCR分析:总的RNA从SER与正常Wistar大鼠整个海马组织中提取,参照Takara试剂盒进行实时定量RT-PCR操作。目的基因Nav1.1、Nav1.3和β₁亚基mRNA的相对表达水平用目的基因与管家基因的平均光密度比值确定。实时定量RT-PCR分析:总的RNA从SER与正常Wistar大鼠整个海马组织中提取,参照Takara试剂盒进行实时定量RT-PCR操作。目的基因七点标准曲线通过反转录产物五倍稀释物进行。目的基因Nav1.1、Nav1.3和β₁亚基mRNA的相对表达水平用目的基因与管家基因的拷贝数比值确定。免疫荧光分析:SER组、正常对照组与新生鼠组（阳性对照）腹腔麻醉后快速取出鼠脑,常规制作冰冻切片。钠通道亚基兔多克隆抗体一抗(anti-Nav1.1,1:25稀释;anti-β_1ex,1:100稀释;anti-Nav1.3,1:25稀释)常温30分钟后4℃过夜,用FITC标记的羊抗兔二抗孵育切片,PBS漂洗三次后,荧光显微镜观察。免疫组化分析:SER组与正常组常规做石蜡切片。钠通道亚基兔多克隆一抗(anti-β_1ex,1:100稀释;anti-Nav1.1,1:25稀释)孵育切片,常温30分钟后4℃过夜。用针对一抗的生物素标记的羊抗兔二抗孵育切片,37℃1小时。DAB显色后,用苏木精做核复染。每只大鼠取10张脑片,其断面水平各鼠均相似,所有脑片均在同一放大倍数（×400）、同一光强度下分析。首先分析海马内平均阳性细胞数,然后用MetaMorph/DP10/BX41图像分析系统测定各组海马CA1、CA3与齿状回蛋白阳性反应物的平均灰度值。免疫印迹分析:SER组、正常对照组与新生鼠组（阳性对照）腹腔麻醉后快速取出鼠海马,提取总蛋白。考马斯亮蓝法测蛋白浓度,上样后进行SDS-PAGE蛋白电泳,一抗(anti-β_1ex,1:500稀释;anti-Nav1.1,1:200稀释;anti-Nav1.3,1:200稀释)孵育。ECL与DAB显影。统计学分析:各组结果以平均值±标准差表示,两组间比较用t检验。结果1、RT-PCR结果显示:SER海马Nav1.1 mRNA的表达显著高于正常对照组（1.14±0.16 in SERs vs 0.68±0.11 in control rats;p＜0.001）;SER海马Nav1.3mRNA的表达亦显著高于正常对照组（0.89±0.17 in SERs vs 0.45±0.16 in controlrats;p＜0.001）;同样的,SER海马β₁亚基mRNA的表达显著高于正常对照组（1.12±0.11 in SERs vs 0.91±0.14 in control rats;p＜0.05）。2、实时定量RT-PCR结果显示:SER海马Nav1.1 mRNA的表达显著高于正常对照组（1.01±0.37 in SERs vs 0.36±0.11 in control rats;p＜0.001）;SER海马Nav1.3mRNA的表达亦显著高于正常对照组（0.55±0.15 in SERs vs 0.39±0.15 in controlrats;p＜0.001）;同样的,SER海马β₁亚基mRNA的表达显著高于正常对照组（0.62±0.13 in SERs vs 0.52±0.07 in control rats;p＜0.05）。综上,SER海马Nav1.1、Nav1.3和β₁亚基在mRNA水平表达上调。3、免疫荧光结果显示:Nav1.1和β₁亚基蛋白在SER和正常大鼠海马各区均有表达;Nav1.3蛋白在新生鼠（三天龄,阳性对照）海马有表达;然而,在正常对照大鼠中未见显著表达Nav1.3蛋白的阳性细胞;值得注意的是,正常对照大鼠微量表达的Nav1.3蛋白在SER海马有显著的阳性表达。4、免疫组化结果证实:Nav1.1和β₁亚基蛋白免疫反应性的阳性细胞在SER和正常大鼠海马各区均有表达,包括CA1、CA3以及齿状回颗粒细胞区;SER海马各区Nav1.1和β₁亚基蛋白免疫反应性的阳性细胞数和平均灰度值均显著高于正常对照大鼠,提示SER海马各区Nav1.1和β₁亚基蛋白表达上调。5、免疫印迹结果进一步证实:SER海马Nav1.1蛋白表达显著高于正常对照组（0.52±0.05 in SERs vs 0.30±0.04 in controlrats;p＜0.001）;SER海马Nav1.3蛋白表达亦显著高于正常对照组（0.38±0.07 in SERs vs 0.17±0.05 in control rats;p＜0.001）;然而,SER海马Nav1.3蛋白表达与新生鼠阳性对照组无统计学差异（0.38±0.07 in SERs vs 0.37±0.05 in neonatal rats;p＞0.05）;SER海马β₁亚基蛋白的表达亦显著高于正常对照组（0.40±0.06 in SERs vs 0.29±0.05 in control rats;p＜0.05）。综上,SER海马Nav1.1、Nav1.3和β₁亚基在蛋白水平表达上调。结论1、首次证实,与正常Wistar大鼠比较,SER海马Nav1.1、Nav1.3和β₁亚基在mRNA水平表达上调。2、首次发现,Nav1.1和β₁蛋白在SER海马各区均有表达,包括CA1、CA3以及齿状回颗粒细胞区。3、首次证实,与正常Wistar大鼠比较,SER海马Nav1.1和β₁亚基在蛋白水平表达上调。4、首次发现,正常成熟的Wistar大鼠微量表达的Nav1.3蛋白在成熟的SER海马有显著的阳性表达。5、SER海马Nav1.1、Nav1.3和β₁亚基在mRNA与蛋白水平表达上调,可能会促进癫痫样兴奋活动的产生,进而构成SER癫痫表型的发作基础。6、SER作为一种良好的癫痫动物模型,可用其筛选钠通道特异性亚型与亚基药物并应用于基因治疗。7、本研究结果在解释遗传性癫痫海马兴奋性的分子机制上有其重要意义。更多还原

【Abstract】 IntroductionVoltage-gated sodium channels（VGSCs）are the primary molecules responsible for the rising phase of action potentials in electrically excitable cells,which consist of a complex of different glycosylated subunits,including a pore-forming a subunit of 260 kDa and fourβsubunits of 32-36 kDa（β₁-β₄ subunit）.Theαsubunit forms the ion-selective pore,inner pore,voltage sensor and inactivation gate,including a transmembrane protein that consists of four homologous domains（Ⅰ-Ⅳ）,each with six transmembrane segments.It is generally agreed that both a andβsubunits play critical roles in modulating the activation and inactivation kinetics of sodium current.In addition,β₁ subunit may function as cell adhesion molecules.Nine mammalian VGSC isoforms have already been identified.In the central nervous system（CNS）,Nav1.1,Nav1.2,Nav1.3 and Nav1.6 are abundantly expressed, and Nav1.1 has been recognized to be highly detectable in the hippocampus, cerebellum,spinal cord,brainstem,neo-cortex,substantia nigra and caudate.Until now, many mutations in VGSC genes have been shown to be critically linked to specific epileptic syndromes.In generalized epilepsy with febrile seizures plus（GEFS⁺）,an autosomal dominant epilepsy syndrome,mutations in the genes coding for VGSCⅠα-isoform（Nav1.1）or;β₁ subunit（SCN1A,SCN1B）have been demonstrated.In human epilepsy,VGSCⅠa-isoform（Nav1.3）mRNA was found to be expressed at significantly higher levels in CA4 hilar cells in the epileptic hippocampus.It has also been reported that increased expression of neonatal Nay 1.3 mRNA was observed 4h after the induction of status epilepticus in neurons of CA1-CA3 and the dentate granule cell layer.Therefore,many previous studies have suggested that genetic abnormalities in the VGSC isoforms predominantly are likely to contribute to neuronal excitability in the CNS.The spontaneously epileptic rat（SER）is a double mutant obtained by mating heterozygous tremor rats（tm）（tm/+）and homozygous zitter rats（zi）（zi/zi）.SER exhibits spontaneous tonic convulsions and absence-like seizures,characterized by simultaneous appearance of 5-7Hz spike-wave complexes in cortical and hippocampal EEG after the age of 8 weeks.The profiles of conventional antiepileptic drugs in SER are quite similar to the efficacy profile in human epilepsy.The mechanism underlying the epileptic seizures in SER was thought to include an abnormality of Ca channel function,an increase in extracellular glutamate concentrations,and enhanced levels of TVacetylaspartate because of lack of the aspartoacylase gene.However,so far,the effects of VGSC in SER have not yet been elucidated.We hypothesized that the etiopathogenisis of SER in genetic epilepsy might be involved in changes of VGSC.Thus,we investigated the expressions of VGSC in SER by researching VGSC Nav1.1,Nay1.3 andβ₁ subunit at the mRNA and protein expression levels of SER,in hope that an insight in their roles in hippocampal excitability could be obtained.Materials and Methods1.MaterialsNormal Wistar rats and SERs at the age of 9-12 weeks were housed in individual cages under a controlled environment（12:12 h light/dark cycle,50-70%humidity, 24℃）.Food and water were available ad libitum.For each experimental method,six SERs were in each experimental group and six Wistar rats were in each control group.The VGSCβ₁ polyclonal antibody generated against the extracellular domain of VGSCβ₁ subunit（KRRSETTAETFTEWTFR）,was a gift from Doctor L.L.Isom, synthesized by the Protein and Carbohydrate Structure Core facility at the University of Michigan.The Nav1.1 and Nav1.3 antibodies were purchased from Sigma.2.MethodsRT-PCR analysis:Total RNA was prepared from the whole hippocampus tissues of SERs（n=6）and control Wistar rats（n=6）using TRIzol（Life Technologies） according to manufacturer’s instructions（Takara）.Real-time RT-PCR analysis:Total RNA was prepared from the whole hippocampus tissues of SERs（n=6）and control Wistar rats（n=6）using TRIzol（Life Technologies）according to manufacturer’s instructions（Takara）.A seven point standard curve for each gene was constructed using five fold serial dilutions of RT product.Immunofluorescence:The brains of SERs（n=6）and Wistar rats（n=6）were dissected out under anesthesia.Neonatal rat hippocampus was tested for Nav1.3 protein analysis and served as a positive control.Tissue sections were incubated with the primary antibody(anti-Nav1.1,1:25 dilution;anti-β_1ex,1:100 dilution;anti-Nav1.3, 1:25 dilution).The sections were incubated at 30 min room temperature and overnight at 4℃,followed by incubation for 30 min in FITC-labeled goat anti-rabbit secondary antibody,and cell nuclei were labeled with Hoechst33258.After washes in PBS,the sections were mounted,viewed and photographed utilizing fluorescence microscopy.Immunohistochemistry:SERs（n=6）and Wistar rats（n=6）were perfused intracardially under anesthesia with normal saline,followed by 4%paraformaldehyde ice-cold fixative.Brains were dissected out and placed in 4%paraformaldehyde for 20 h at 4℃.The primary antibodies(anti-β_1ex,1:100 dilution;anti-Nav1.1,1:25 dilution) were incubated for 30 min at room temperature and overnight at 4℃,and they were carried out using avidin-biotin peroxidase method and 3,3′-diaminobenzidine（DAB） as a chromogen.Control sections incubated without the primary antibody or with pre-immune sera were performed.The intensity,the cellular localization,and the frequency of immunoreactive cells were examined in different hippocampal regions （CA1,CA3 and dentate gyrus）of control rats and SERs.The expression levels of Nav1.1 andβ₁ subunit were quantified by counting immunoreactive cells and measuring the intensity of staining using a computerized image analysis system.Western blot analysis:Western blot analysis was performed on samples of the whole hippocampus of adult SERs（n=6）and adult Wistar rats（n=6）.For Nav1.3 protein analysis,six neonatal rats were served as positive controls.Samples were homogenized in lysis buffer containing 10 mM Tris（pH 8.0）,150 mM NaCl,10% glycerol,1%NP-40,5 mM ethylenediamine tetra-acetic acid（EDTA）and protease inhibitor cocktail.Samples were incubated over night in TTBS:3%BSA,0.1%sodium azide,containing the primary antibodies(anti-β_1ex,1:500 dilution;anti-Nav1.1,1:200 dilution;anti-Nav1.3,1:200 dilution).Immunoreactive bands were visualized using DAB and ECL kits.The levels of Nav1.1,Nav1.3 andβ₁ protein were evaluated by measuring optical densities of the protein bands using Scion Image for Windows image-analysis software.Statistical analyses:Mean and standard deviation were calculated for all measurements in this study.Student’s t-test was performed to determine statistical significance,which was evaluated at p＜0.05.All statistical analyses were conducted using SPSS for Windows version 12.0.Results1.The mRNA expressions of VGSC Nav1.1,Nav1.3 andβ₁ subunit in SERs and control rats hippocampusThe data suggested that the mRNA expression of Nav1.1 in SERs hippocampus was significantly higher than that of control groups（1.01±0.37 in SERs vs 0.36±0.11 in control rats;p＜0.001）by real-time RT-PCR;The mRNA expression level of Nav1.3 was abundantly increased compared with the control rats（0.55±0.15 in SERs vs 0.39±0.15 in control rats;p＜0.001）;The mRNA expression ofβ₁ subunit was also higher than that of control groups in hippocampus（0.62±0.13 in SERs vs 0.52±0.07 in control rats;p＜0.05）.The data by real-time RT-PCR were in agreement with the data by means of RT-PCR.On the whole,the mRNA expressions of VGSC Nav1.1,Nav1.3 andβ₁ subunit were up-regulated in SERs hippocampus.2.The localization expressions of VGSC Nav1.1,Nav1.3 andβ₁ subunit in SERs and control rats hippocampusNav1.1 andβ₁ protein in SERs and control tissue were localized widely in the hippocampus by immunofluorescence.Under our investigation,Nav1.3 protein that was expressed in the neonatal（at the age of 3 days）hippocampus,was considered as a positive control.It was noting that the clear Nav1.3-immunoreactive cells were not found in control adult Wistar rats hippocampus.However,Nav1.3 protein that was barely detected in adult Wistar rats was found to be expressed abundantly in adult SERs hippocampus.3.The protein expressions of VGSC Nav1.1 andβ₁ subunit in SERs and control rats hippocampusBy means of immunohistochemistry,the protein expressions of Nav1.1 andβ₁ subunit in the hippocampus of SERs were significantly higher than those in control rats. In addition,the increases in Nav1.1 andβ₁ staining were clearly present throughout the hippocampus of SERs,including CA1,CA3 and dentate gyrus regions.Taken together, the data showed that the expressions at the protein level of Nav1.1 andβ₁ subunit were up-regulated in SERs hippocampus.4.Additional tests that support the protein expressions of VGSC Nav1.1,Nav1.3 andβ₁ subunit in SERs and control rats hippocampusThe protein expression of Nav1.1 in SERs hippocampus was significantly higher than that in control groups by western blot analysis（0.52±0.05 in SERs vs 0.30±0.04 in control rats;p＜0.001）;The protein expression level of Nav1.3 was abundantly increased compared with the control rats（0.38±0.07 in SERs vs 0.17±0.05 in control rats;p＜0.001）.However,it seemed unchanged compared with neonatal rats（0.38±0.07 in SERs vs 0.37±0.05 in neonatal rats;p＞0.05）.Taken together with the data of immunofluorescence,Nav1.3 protein that was barely detected in adult Wistar rats was found to be expressed abundantly in adult SERs hippocampus.The protein expression ofβ₁ subunit in SERs was higher than that of control rats in hippocampus（0.40±0.06 in SERs vs 0.29±0.05 in control rats;p＜0.05）.Altogether,the findings using western blot analysis further verified up-regulation expressions of VGSC Nav1.1,Nav1.3 andβ₁ subunit at the protein level in SERs hippocampus.Conclusions1.The mRNA expressions of Nav1.1,Nav1.3 andβ₁ subunit were first detected to be up-regulated in SERs hippocampus compared with control Wistar rats.2.Nav1.1 andβ₁ subunit were first observed to be localized widely in SER hippocampus including CA1-CA3 and DG.3.The protein expressions of Nav1.1 andβ₁ subunit were first found to be up-regulated in SERs hippocampus compared with control Wistar rats.4.Nav1.3 protein that was barely detected in adult Wistar rats was found to be expressed abundantly in adult SERs hippocampus.5.Sodium channel Nav1.1,Nav1.3 andβ₁ subunit up-regulation at the mRNA and protein levels of SER hippocampus might contribute to the generation of epileptiform activity and underlie the observed seizure phenotype in SER.6.The SER can be used as an epileptic animal model to screen effective specific VGSC isoforms for medicine and gene therapy.7.The results of this study may be of value in revealing components of the molecular mechanisms of hippocampal excitation that are related to genetic epilepsy.更多还原