【作者】 郝勇；

【导师】 洪震； 任惠民； 丁玎； 朱国行；

【作者基本信息】 复旦大学 ， 神经病学， 2007， 博士

【摘要】 癫痫是神经系统常见的临床综合征，慢性、突发性和反复刻板发作为其主要临床特点。尽管新抗癫痫药(antiepileptic drugs，AEDs)不断问世，但仍有30％患者的发作不能控制，成为难治性癫痫。这部分患者多为复杂部分性发作(多见于成人)和各种癫痫综合征(多为儿童)，不仅对常规AEDs的反应较差(具有耐药性)，即使是新型AEDs对这部分癫痫患者的疗效也不十分肯定。大量研究表明由于癫痫源性脑组织的血脑屏障(blood-brain barrier，BBB)上具有药物外排作用的多药转运体(multidrug transporters，MDTs)的过度表达，限制了脑组织对AEDs的摄取，影响了药物到达作用靶点而形成耐药。癫痫发作可引起MDTs的过度表达，癫痫发作又引起谷氨酸释放增加，谷氨酸通过活化NMDA受体上调大鼠脑微血管内皮细胞P-gp的表达，提示癫痫发作后MDTs的过度表达可能与NMDA受体的活化有关，我们首先观察边缘叶癫痫发作大鼠模型癫痫持续状态(status epilepticus，SE)后72 h内海马MDTs的表达变化；然后分别在SE后MDTs mRNA和蛋白表达最高时点观察NMDA受体拮抗剂MK801对MDTs表达的影响；最后在细胞水平观察谷氨酸刺激对大鼠脑微血管内皮细胞(rat brain microvessel endothelial cells，RBMECs)mrp2 mRNA表达和外排功能的影响，以及MK801预处理后对谷氨酸作用的影响。第一部分多药转运体在边缘叶癫痫发作后的动态表达变化目的：探讨SE终止后72 h内P-gp、Mrp2、Bcrp表达的时间规律，明确MDTs表达的细胞类型分布。材料与方法：制备氯化锂—匹罗卡品癫痫发作大鼠模型，用实时荧光定量RT-PCR法检测SE终止后0h、3h、6h、24h、72h和control组海马组织P-gp、Mrp2、Bcrp、Ralbpl、Pxr的mRNA表达；Western blot检测SE终止后3h、6h、24h、72h和control组海马组织P-gp、Mrp2、Bcrp的蛋白表达；免疫荧光检测SE终止后24h和对照组P-gp、Mrp2、Bcrp分别与vWF、MAP2、GFAP双标染色，以上各组每组6只大鼠。结果：SE终止后72h内P-gp、Mrp2、Bcrp mRNA和蛋白表达均先升高后降低，3种MDTs mRNA表达均在SE终止后6h最高(6h时mdr1b mRNA的平均拷贝数为36148.8，3种MDTs mRNA的表达与control相比，P＜0.01)，蛋白表达均在24 h时最高(Mrp2平均相对光密度值为0.27，3种MDTs与control相比，P＜0.01)，Pxr mRNA的表达在3 h时最高(平均mRNA拷贝数为136066.7，与control相比，P＜0.01)。正常对照组3种MDTs均只在毛细血管内皮细胞表达，SE终止后24 h，3种MDTs在神经元和星形胶质细胞上亦有少量表达。结论：癫痫发作诱导P-gp、Mrp2、Bcrp暂时性过度表达，癫痫发作可诱导神经元和星形胶质细胞表达以上3种MDTs。第二部分谷氨酸NMDA受体拮抗剂MK801对癫痫发作后多药转运体P-gp、Mrp2、Bcrp表达的影响目的：观察NMDA受体对边缘叶癫痫大鼠发作后P-gp、Mrp2、Bcrp表达的影响，探讨NMDA受体是否能对癫痫发作后上述3种MDTs的表达进行调控。材料与方法：动物共分为3组：control、MK801和SE组，SE组即氯化锂—匹罗卡品癫痫发作大鼠模型组，MK801组即SE组加MK801(0.5 mg／kg)预处理组，control组即除匹罗卡品改用生理盐水代替外，其他给药同SE组。使用实时荧光定量RT-PCR和Western blot分别检测P-gp、Mrp2、Bcrp mRNA(SE终止后6 h)和蛋白(SE终止后24 h)表达，使用免疫组化检测SE终止后24 h时上述3种MDTs的表达分布。结果：SE组P-gp、Mrp2、Bcrp mRNA和蛋白表达均较control组明显升高(与control组相比，除mdr1a P＜0.05，其他均为P＜0.01)，MK801组上述MDTs的表达均较SE组明显下降，MK801组除Mrp2蛋白表达仍明显高于control组外，其他MK801各组mRNA和蛋白表达均与control组无统计学差异。免疫组化显示：SE组边缘系统脑区和大脑皮层P-gp的表达(阳性面积百分比和累积光密度值)明显高于control组和MK801组，后两者无统计学差异；除杏仁核外，SE组其他边缘系统和大脑皮层Mrp2的表达较control组明显增高(阳性面积百分比)，MK801组除梨状皮质明显高于control组外，其他脑区与control组相比无统计学差异；SE组边缘系统脑区和大脑皮层Bcrp的表达明显高于control组，除海马齿状回区MK801组较SE组Bcrp表达无差异外，MK801组其他脑区Bcrp的表达较SE组明显下降，并且MK801组与control组各脑区Bcrp的表达无差异。结论：NMDA受体拮抗剂MK801能逆转癫痫发作后P-gp、Mrp2、Bcrp的过度表达，NMDA受体的活化参与介导癫痫发作后P-gp、Mrp2、Bcrp的过度表达。在边缘脑区和大脑皮层中，SE组上述3种MDTs表达较control组明显增加，大多MK801组明显低于SE组，并与control组表达水平相当。第三部分谷氨酸、NMDA受体拮抗剂MK801对大鼠脑微血管内皮细胞mrp2 mRNA表达和外排功能的影响目的：观察谷氨酸对RBMECs上mrp2 mRNA表达和外排转运功能的影响，探讨NMDA受体是否影响谷氨酸刺激RBMECs后mrp2 mRNA和外排转运功能的变化。材料与方法：分离2周龄SD大鼠的大脑皮层微血管段，并进行原代RBMECs培养。待RBMECs生长至融合后，进行以下实验。MTT法检测不同浓度谷氨酸刺激对RBMECs活力的影响，细胞分组：control组，不给任何特殊处理；Glu组，给100μM谷氨酸刺激30 min后吸去上清，再培养24 h；MK801组，加入谷氨酸前，用100μM MK801预处理15 min，其他处理同Glu组。实时荧光定量RT-PCR检测mrp2 mRNA的表达。底物外排实验检测不同干预对RBMECs上Mrp2外排转运功能的影响。结果：MTT法结果显示300μM及其以上浓度的谷氨酸可明显引起RBMECS活力的下降，选择相对浓度较高，但又无明显影响RBMECs活力的谷氨酸浓度100μM，实时荧光定量RT-PCR结果显示，100μM谷氨酸刺激后，RBMECs上mrp2 mRNA的表达明显高于control组和MK801组(P＜0.05)，control组和MK801组比较无差异(P＞0.05)。底物外排实验结果表明Glu组胞外／胞内荧光强度比值明显高于control组和MK801组(分别为P＜0.05)，MK801组与control组比较无统计学差异(P＞0.05)。结论：谷氨酸刺激RBMECSs可诱导mrp2 mRNA表达和外排转运功能的增强，NMDA受体的拮抗剂可完全逆转谷氨酸的这一作用。结论1、癫痫发作诱导P-gp、Mrp2、Bcrp暂时性过度表达，癫痫发作可诱导神经元和星形胶质细胞表达以上3种MDTs。2、NMDA受体拮抗剂MK801能逆转癫痫发作后P-gp、Mrp2、Bcrp的过度表达，NMDA受体的活化参与介导癫痫发作后P-gp、Mrp2、Bcrp的过度表达。3、谷氨酸刺激RBMECSs可诱导mrp2 mRNA表达和外排转运功能的增强，并且NMDA受体的拮抗剂MK801可以逆转谷氨酸的这一作用。更多还原

【Abstract】 Epilepsy is a common clinical syndrome in nervous system, with chronic, accidental,and repeatedly stereotyped as its primary clinical characteristics. Although the therapyfor Epilepsy is advancing, there are still about 30% of all Epilepsy patients, withseizure out of controlled, belonging to refractory epilepsy including complex partialseizures (most seen in adults) and various kinds of epilepsy syndrome (most seen inchildren). Not only they are failed to response to the routine antiepileptic drugs(AEDs) (drug resistance), but also the new AEDs have uncertain effect for them. A lotof studies indicated that the overexpression of Multidrug Transporters (MDTs) on theblood brain barrier (BBB) of epileptogenic brain tissue limited brain uptake AEDs,interfered with AEDs attaining the taget, and developed drug resistance in the end.Seizure could induce the overexpression of MDTs, and it also cause glutamate torelease more. And glutamate could upregulate P-gp’s expression by activating NMDAreceptor in the rat brain microvessel endothelial cells (RBMECs), which indicated thatthe overexpression of MDTs during seizure has something to do with the activation ofNMDA receptor. Thus, firstly, we observe the expression of MDTs in hippocampus inthe limbic seizure rat model within 72 h after status epilepticus (SE) was terminated.Secondly, we observe the effects of NMDA receptor antagonist, MK801, on theexpression of MDTs’ mRNA and protein respectively at the peak time of their mRNAand protein expression within 72 h after SE was terminated. Thirdly, we explored theeffects of glutamate on Mrp2’s mRNA expression and efflux function, and the effectsof MK801’s pretreatment on the changes of Mrp2’s mRNA expression and effluxfunction of RBMEC caused by glutamate in vitro.PartⅠThe Expression of MDTs after Limbic SeizureAim: To explore the time expression pattern of P-gp, Mrp2, and Bcrp within 72 hafter SE was terminated, and to determine the cell pattern distribution of these MDTs’expression. Methods: Limbic seizure model was induced by lithium chloride andpilocarpine. Realtime fluorescent quantitative RT-PCR (RT-qPCR) was employed todetermine P-gp, Mrp2, and Bcrp mRNA expression in hippocampus at 0h, 3h, 6h,24h, 72h after SE was terminated and of control group. Protein expression of P-gp, Mrp2, and Bcrp in hippocampus was determined by Western blot at 3h, 6h, 24h, 72h after SE was terminated and of control group. Brain slices were double labeled byP-gp, Mrp2, and Bcrp with vWF, MAP2, and GFAP respectively by Immunofluerence.Each group mentioned included 6 rats. Results: mRNA and Protein expression ofthese MDTs increased instantly after SE was terminated, mRNA expression of eachreached peak at 6h, while their protein expression attained peak at 24h. It is at 3hthat pxr mRNA expression reaches its peak. The expression of P-gp, Mrp2, and Bcrplocated on endothelial cells of brain capillary in control group, while their expressionincreased and appeared on a few of neurons and astrocytes at 24 h after SEterminated.Conclusion: Seizure induced transient overexpression of P-gp, Mrp2, and Bcrp. Alsoseizure could induce these MDTs’ expression in neurons and astrocytes.PartⅡThe Effects of Glutamate NMDA Receptor Antagonist. MK801, on the Expression of P-gp, Mrp2 and Bcrp after SeizureAim: To observe the effects of NMDA receptor on P-gp, Mrp2, and Bcrp expressionafter limbic seizure, and to explore whether NMDA receptor could regulate theexpression of these MDTs. Methods: Rats were divided into 3 groups: SE group wasrefered to the limbic seizure rat model induced by LiCl-Pilocarpine; MK801 groupwas pretreatmented with MK801 (0.5 mg/kg); control group was given Normal Salineinstead of pilocarpine, and other treatment of MK801 and control group were thesame as SE group. P-gp, Mrp2, and Bcrp’s mRNA and protein expression weredetermined by RT-qPCR (6h after SE was terminated) and Western blot (24 h afterSE was terminated) respectively. Immunohistochemistry was employed to determinethe expression distribution of P-gp, Mrp2 and Bcrp. Results: The expression of P-gp,Mrp2 and Bcrp in SE group were significantly higer than control group, while theexpression of these MDTs in MK801 goup were lower than SE group. Exception forthe protein expression of Mrp2 in MK801 group, there was no difference between theexpression of these MDTs in MK801 and control group. Immunhistochemistry resultsshowed that the expression of these MDTs in almost each limbic brain area in SEgroup were much higher than MK801 and control group. Conclusion: NMDAreceptor antagonist, MK801, could reverse the overexpression of P-gp, Mrp2 andBcrp induced by seizure. The activation of NMDA receptor might be involved in theoverexpression of P-gp, Mrp2, and Bcrp during seizure. PartⅢThe Effects of Glutamate, and NMDA Receptor antagonist, MK801, on mRNA expression and efflux function of Mrp2 in RBMECsAim: To observe the effects of glutamate on the mRNA expression and the effluxfunction of Mrp2, and to explore whether NMDA receptor has an effect on thechanges of expression and function of Mrp2 induced by glutamate. Methods: Isolatedmicrovessel segments from the cortex of 2-week-old rats, and rat brain microvesselendothelial cells (RBMECs) primary culture was carried out. After RBMECs becameconfluent, we undertook the following experiments. The cells were divided into 3groups: control, Glu, and MK801 group. The cytotoxicity of different concentration ofglutamate on RBMECs was determined by MTT to identify an appropriateconcentration for glutamate intervention, mRNA expression of mrp2 was determinedby RT-qPCR. And Mrp2 efflux function was determined by substrate effluxexperiment. Results: MTT results showed that glutamate at 300μM or above causedsignificant cytotoxicity, therefore we selected 100μM as the glutamate concentrationfor intervention. RT-qPCR results demonstrated that the mRNA expression of mrp2 inGlu group was significantly higher than MK801 and control group (P＜0.05). And theefflux experiments showed that the ratio of extracellular/intracellular concentration offluorescent substrate was higher in Glu group than MK801 and control group(P＜0.05). Conclusion: Glutamate could induce the upregulation of mrp2 mRNAexpression and the reinforcement of Mrp2’s efflux function. Furthermore, NMDAreceptor antagonist, MK801, could reverse these effects of glutamate.Summary1. Seizure induces transient overexpression of P-gp, Mrp2, and Bcrp. Furthermore,seizure could induce these MDTs’ expression in neurons and astrocytes.2. NMDA receptor antagonist, MK801, could reverse the overexpression of P-gp,Mrp2 and Bcrp induced by seizure. The activation of NMDA receptor might beinvolved in the overexpression ofP-gp, Mrp2, and Bcrp during seizure.3. Glutamate could induce the upregulation of mrp2 mRNA expression and thereinforcement of Mrp2’s efflux function. Furthermore, NMDA receptorantagonist, MK801, could reverse these effects of glutamate.更多还原