【作者】 刘昕；

【导师】 肖波；

【作者基本信息】 中南大学 ， 神经病学， 2010， 硕士

【摘要】 目的：观察大鼠癫痫持续状态(SE)后3天内海马组织内NKCC1及GABAARα1表达的改变,及使用BDNF中和性抗体血清(anti-BDNF)干预后其表达的变化,探讨NKCC1在SE中可能机制及其与GABAARα1的关系,以及BDNF在SE发生过程中对NKCC1和GABAARα1表达的影响,研究其在癫痫发病机制中的作用,从而寻找癫痫新的治疗靶点。方法：健康成年雄性SD大鼠135只,随机分为生理盐水对照组(15只),SE组(60只)和干预组(60只)。用氯化锂-匹罗卡品(LiC1-PILO)腹腔注射制作大鼠SE模型(SE组),在LiC1-PILO造模前3～4小时立体定向侧脑室注射anti-BDNF制作干预组模型(干预组),两组均选取SE后2h,6h,24h,3d为实验点。观察两组大鼠在SE诱发过程中行为学差异,RT-PCR检测各组大鼠海马NKCC1、GABAARα1 mRNA表达改变,Western-Blot检测NKCC1、GABAARα1蛋白的表达,利用免疫组化检测海马各区NKCC1、GABAARα1时空分布的特点。结果：1.动物实验结果：与SE组相比,干预组SE诱发成功率低(分别为90.7%和75.27%),SE诱发成功后生存状态好,死亡率低(分别为23.08%和14.29%)。与SE组相比,干预组诱发SE所需PILO平均用量(分别为22.78±6.69mg／kg和42.78±6.69mg／kg)、诱发SE所需的平均时间(分别为41.17±18.94min和89.72±11.28min)及10%水合氯醛总量平均值(分别为0.91±0.05ml／100g和0.76±0.06ml／100g)均有明显差异,具有统计学意义(p<0.01)。2.NKCC1表达改变：SE组NKCC1的mRNA及蛋白在SE后均高于对照组(p<0.01),2h显著上调,6h达最高,24h后开始恢复,3d时仍高于正常。以上各时间点海马NKCC1蛋白表达亦均高于对照组(p<0.01),在海马不同亚区,在SE后两小时开始,NKCC1在CA1及CA3区均显著增高。干预组NKCC1的mRNA及蛋白表达在2h显著增加,6h达最高,24h后开始明显恢复,3d时接近正常。干预组2h、6h、24h和3d蛋白含量与对照组相比差异显著,具有统计学意义(p<0.01)。NKCC1蛋白在海马各区的时空分布趋势与SE组相似。干预组与SE组相比,SE后各时间点NKCC1的表达差异非常显著(p<0.01)。3. GABAARα1表达改变：SE组GABAARα1的mRNA及蛋白在SE后2h-6h表达显著下调(p<0.01),24h继续下降至最低值(p<0.01),至3d突然增高(p<0.01)。GABAARα1在海马不同亚区表达SE后2h开始CA1区及CA3区的GABAARα1蛋白表达显著进行性下调。干预组GABAARα1的mRNA及蛋白表达在SE后2h达最低值,6h后上调,3d达最高值(p<0.01),GABAARα1蛋白表达在海马的空间分布特点与SE组相似。对照SE组,干预组GABAARα1蛋白表达变化的幅度小,周期短,两组GABAARα1的表达在SE后各时间点的差异显著,均具有统计学意义(p<0.01)。结论：1.NKCC1蛋白表达在SE后的上调可能是SE后GABAAR介导的兴奋性作用产生的重要机制之一。2. Anti-BDNF干预可通过抑制BDNF的表达,延缓癫痫发生并减弱癫痫发作的严重程度。更多还原

【Abstract】 ObjectiveIn this study, we want to observe the expression of NKCC1 and GABAARal mRNA and protein in rat hippocampus 3 days after status epilepsy(SE) and the effect of using of sheep BDNF neutralization antiserum (anti-BDNF). We want to investigate the role of NKCC1 in the formation of SE, and the relationship between NKCC1 and GABAARal, and demonstrate the effect of BDNF on modulating the activiation and expression of NKCC1 and GABAARal in SE, and discuss the function of them in epileptic pathogenesis in order to search new target of anti-epileptic treatment.Method130 SD male rats are randomly divided into N.S control group(15 rats), SE group(60 rats) and experiment group(60 rats). Rats of SE group are induced SE by LiC1-PILO i.p. Rats of experiment group received the same treatment as SE group, except for being injected with anti-BDNF 4h before PILO i.p. Rats of both SE group and experiment group are randomly divided into 6 subgroups respectively(15 rats each), and are sacrificed 2h,6h,24h,3d after SE ended. The changes of ethology of two groups during and after SE induced are observed and evaluated. The expression and distribution of NKCC1 and GABAARal in hippocampus are detected with RT-PCR, immunohistochemisty and Westem Blot.Results1. Results of animal model:Compared with the SE group, the achievement ratio of experient group(respectively 90.7% and 75.27%), and mortality rate are lower than SE group (respectively 23.08% and 14.29%). Compared with SE group, the differences of mean dosage of PILO of experiment group for inducing SE(respectively 22.78±6.69mg/ kg and 42.78±6.69mg/kg), mean time needed to induce SE(respectively 41.17±18.94min and 89.72±11.28min), and mean dosage of 10% Chloral Hydrate for ending SE(respectively 0.91±0.05ml/100g and 0.76±0.06ml/ 100g) have significant statistic differences(p<0.01).2. Expression of NKCC1 mRNA and protein are acute substantially up-regulated in SE group within 3d after SE ended, they increase at 2h after SE ended, and come to peak at 6h,then they gradually and slowly recover at 24h, they remain higher than normal at 3d. There are significant statistic differences in all four time points when compared with control (p<0.01). Up-regulation of NKCC1 significantly appears in CA1 and CA3 area after SE. Those of experiment group is with similar distribution but they almost near the normal at 3d after SE ended. Compared with SE group, expression of NKCC1 of the experimental group are significant statistic different in all detected time points(p<0.01).3. The GABAARal mRNA and protein expression of SE group is down-regulated within 24h, but greatly up-regulated at 3d after SE, with significant statistic differences compared with control(p<0.01). The space distribute pattern of GABAARal is great distinct between different areas. It is acute substantially decreased in CA1 and CA3 area since 2h. Compared with control, the GABAARal expression of experiment group is down-regulated at 2h, but up-regulated from 6h to 3d(p<0.01). The distribution pattern of GABAARal of experiment group is similar with SE group. There are significant statistic difference when GABAARal expression of experiment group is compared with SE group at 6h-3d after SE(p<0.01).Conclusion1. The up-regulation of NKCC1 may play an important role in GABAAR-mediated excitation after SE.2. The treatment of anti-BDNF can decrease the action of BDNF protein, delay the onset of SE and reduce the degree and duration of severe SE.更多还原