【作者】 杜娃；

【导师】 蔡际群；

【作者基本信息】 中国医科大学 ， 药理学， 2004， 硕士

【摘要】 目的 电压门控性钠通道α亚基的变化与癫痫发病机制之间存在着密切的联系，本研究以遗传性癫痫动物模型自发性癫痫大鼠为实验对象，通过比较自发性癫痫大鼠和Wistar大鼠脑内电压门控性Ⅰ、Ⅱ和Ⅲ型钠通道α亚基mRNA的相对含量，寻找遗传性癫痫发病机制与钠通道编码基因之间存在的关系，为癫痫的基因治疗提供理论依据。 方法 自发性癫痫大鼠及Wistar大鼠每组5只，将大鼠断头处死，迅速取脑，液氮速冻，切成400μm的冠状切片，显微镜下观察后在皮质、齿状回、海马CA1和CA3区域进行针孔取样，制备这四个区域组织的总RNA。用逆转录酶PCR试剂盒逆转录制备cDNA后，分别施行PCR方案1、2和3对制得的模板cDNA进行特异性的扩增。对PCR方案1扩增得到产物进行琼脂糖凝胶电泳，紫外扫描分析比较自发性癫痫大鼠及Wistar大鼠脑内四个区域电压门控性钠通道α亚基mRNA的相对含量。对PCR方案2和方案3扩增得到的产物进行限制性内切酶酶切分析，Msp Ⅰ切割Ⅰ型钠通道，BamHⅠ切割ⅡA亚型钠通道，Sfu Ⅰ切割ⅡN亚型钠通道，Dra Ⅰ切割Ⅲ型钠通道，Nde Ⅰ切割ⅢA亚型钠通道，Sac Ⅰ切割ⅢN亚型钠通道。对酶切产物分别进行琼脂糖凝胶电泳，紫外扫描分析检测Ⅰ型、ⅡA亚型、ⅡN亚型、Ⅲ型、ⅢA亚型和ⅢN亚型钠通道的表达情况。统计分析采用学生氏t检验。结果 自发性癫痛大鼠脑内皮质、齿状回和海马CAI区域内钠通道mRNA的相对含量略高于Wistar大鼠，但经分析无显著统计学差异(P>0.05，P值分别为0.36、0.24和0.41)，CA3区域内钠通道mRNA相对含量与Wistal大鼠相同。经特异性的限制性内切酶MsPI、BaxnHI、Sful和Dral消化后，自发性癫痈大鼠脑内I型，nA亚型，llN亚型及111型钠通道在整个钠通道中所占的百分比也与Wistar大鼠无显著差异。l型钠通道扩增产物经限制性内切酶Sacl和Ndel切割，结果显示IA与IllN亚型的电泳片段同时出现在自发性癫痛大鼠脑内，紫外分析标明自发性癫痛大鼠脑内lllN亚型钠通道mRNA明显增高(P<0.01)。讨论 一般说来班A亚型钠通道在正常Wistar大鼠脑高表达，其分布及表达与nI型钠通道分布和表达情况一致。而IllN亚型钠通道仅在胚胎期高表达，所以成熟大鼠脑内基本观察不到IllN亚型钠通道的出现。由SER脑内lllN亚型钠通道表达明显增高，我们可以推断sER脑内111型钠通道基因编码的两种外显子(A和N)互斥剪接的调控受到了影响。由于111型钠通道的A亚型与N亚型的构成比例发生了变化，我们推测其第I同源结构域内200号的天冬氨酸被丝氨酸取代，进而造成细胞外表面电荷数量发生相应的改变。电压性门控是指对应着电压变化时发生的通道传导和非传导状态的改变，电压门控性通道随着电压的变化其构象也会发生改变，因而如果带负电荷的天冬氨酸被中性氨基酸丝氨酸大量取代，可导致细胞膜外表面负电荷减少，正电荷相对增加，而且其发生改变的氨基酸又恰好接近电压感受器，因而产生了一系列的钠通道被激活的改变，比如钠通道的开放数目增加或开放频率增快，这些变化都可导致神经元过度放电，与癫痛的电生理改变恰好吻合，但需要做进一步的电生理研究来证实我们的这种推测。结论 1.本研究采用PCR方案1扩增后，该段钠通道序列范围内SER与Wistar大鼠的扩增产物片段大小相同，标明该段编码区域内SER没有发生大的基因缺失或突变。 2.PCR方案2和3的扩增产物经限制性内切酶酶切消化后，sER脑内电压门控性I、nA、llN和111型钠通道的表达和分布与Wistal大鼠相比无显著差异，nA与nN外显子的互斥剪接未受到影响，但是IA与nIN的外显子互斥剪接发生了改变，lllA与lllN亚型钠通道同时出现于成熟SER脑内。 3 .IN亚型钠通道在SER脑内表达明显增高，这一改变可能会导致电压依赖性门控和电压门控性钠通道对钠离子的通透性均发生改变，进而产生一系列的钠通道被激活的变化。更多还原

【Abstract】 The relationship between the change of voltage - gated sodium channel a subunit and pathogenesis of epilepsy is very closely. This report used genetic epileptic animal model, spontaneously epileptic rat ( SER) , as experimental animal , and compared voltage - gated I , II and III sodium channel a subunit mRNA relative amount of SER with that of normal Wistar rats in brain. To find the exact relationship between genetic epilepsy pathogenesis and encoding voltage - gated sodium channel sequence, and provide theoretical foundation for gene therapy of epilepsy.MethodsFive SER and Wistar rats were in each group. They were killed by decapitation , and the brain were removed rapidly, frozen in liquid vapors and spliced into 400 um coronal section. Under binocular microscopic needles punches were sampled from four different regions: cortex, dentate gyrus, hippocampus CA1, and CA3. Total RNA extracted from these four regions tissue, with RNA PCR kit performed PCR protocol 1, 2 and 3, respectively. Amplified products according to PCR 1 protocol were run agarose gel electrophoresis, with ultraviolet scanning analysis compared voltage - gated sodium channel a subunit mRNA relative amount of SER with that of Wistar rats in four different regions of brain. Amplified products according to PCR 2 and/or 3 were performed specific restriction enzyme analysis, Msp I for NaCh I , BamH I for NaCh II A, Sfu I for NaCh II N, Dra I for NaCh III, Nde I for NaCh III A, and Sac I for NaCh III N, respectively. Digested products were run agarose gel electrophoresis and with ul-traviolet scanning detected the change of subtype I , IIA, II N, III A, and III N.ResultsSER all types of sodium channel expressed a little higher than control groups in cortex, dentate gyrus and CA1, but had no significant difference ( P> 0.05, P value is 0.36, 0. 24, and 0.41, respectively ) , and were the same with control rats in CA3. Digested by specific restriction enzyme, SER type I ,II A, II N, and III sodium channel percentage constitution in the whole sodium channel are all similar to Wistar rats. Specific amplified products for type III sodium channel under restriction analysis, showed that III A and III N fragments were present in SER brain simultaneously. But during brain development, subtype III N expressed highly in embryonic stage, and disappeared postnatal 10 days, subtype III A replaced it gradually. So III N should not have appeared in a-dult Wistar rats. These indicated that SER subtype IIIN mRNA increased significantly (P < 0.01).ConclusionsIn the amplified voltage - gated sodium channel a subunit coding sequences , there was no large deletion or mutation in SER brain. But mRNA mutually exclusive splicing of type III sodium channel two exons, III A and III N, in SER brain was not regulated correctly during brain development. This might result in discharging excessively by neuron, but it required the evidence verified by voltage - gated sodium channel electrophysiological studies.更多还原