【作者】 于臻；

【导师】 孙若鹏；

【作者基本信息】 山东大学 ， 小儿内科， 2008， 硕士

【摘要】 研究背景癫痫是一种反复发作的、慢性脑部疾病。尽管近几年来诊断和治疗水平有了很大提高,但仍有1/4以上的患者不能控制发作,临床上称为难治性癫痫（RE）。目前发现难治性癫痫的主要发病机制之一是多药耐药基因及其表达产物P-gp蛋白在难治性癫痫脑组织中的过度表达。左乙拉西坦作为一种新型的抗癫痫药物,对MDR1表达的影响目前尚不明了。利用海人酸制备的慢性癫痫模型是一种接近于人类颞叶癫痫的模型,而颞叶癫痫是一种较常见的难治性癫痫。因此制备海人酸致癫模型来研究左乙拉西坦对MDR1的影响对指导临床有重要意义。氟桂利嗪是一种钙离子拮抗剂,据报道在动物实验中对多药耐药基因有逆转作用,对难治性癫痫辅助治疗有效,但目前还未在临床中常规应用。氟桂利嗪对难治性癫痫治疗的作用机制尚不明了,而且能否在儿科中应用及疗效还需探讨。因此,通过检测难治性癫痫患儿外周血MDR1 mRNA的表达,以及对氟桂利嗪的反应和耐受情况来观察氟桂利嗪作为儿科辅助用药的可能性具有重要意义。目的探讨左乙拉西坦对多药耐药基因的影响;研究难治性癫痫患儿外周血中MDR1 mRNA的表达及氟桂利嗪作为辅助用药在小儿难治性癫痫治疗中的作用及耐受性。方法1、左乙拉西坦对MDR1表达的影响:选用生后7d（P7）的Wistar大鼠70只,随机分为癫痫观察组38只和正常对照组32只。癫痫观察组给予海人酸（kainicacid,KA）1mg/kg（0.5ml/kg）,腹腔注射致痫,对照组应用相同方法给予相同剂量的生理盐水。待慢性癫痫模型建立后,癫痫观察组随机分为慢性癫痫（简称EP）观察组,13只,慢性癫痫治疗（简称EP+LEV）组,15只;正常对照组分为生理盐水对照（control）组,16只和正常生理盐水对照治疗（control+LEV）组,16只。治疗组给予左乙拉西坦（80mg/kg）灌胃,治疗8w后,断头取海马,称重,一步法逆转录—聚合酶链反应（PT-PCR）检测mdr1a和mdr1b mRNA的表达。2、应用RT-PCR半定量检测86例受试者（难治性癫痫患儿64例,其中氟桂利嗪治疗组36例,安慰剂组28例;正常对照组22例）外周血MDR1 mRNA的表达情况。辅助治疗7～8w（氟桂利嗪治疗组给予5mg,po,qⁿ;安慰剂组给予维生素B₆10mg,po,qⁿ）,进行临床疗效观察。发作减少≥50%为有效,用药后复查MDR1 mRNA的表达。同时观察患儿对药物的反应和耐受性。结果1、注射海人酸后2～10min出现5—7级发作,平均潜伏时间为（4.3±3.1）min,第2～3w起出现自发性反复惊厥（spontaneous recurrent seizure,SRS）,每日发作约4～5次。Wistar大鼠共死亡8只,造模失败2只,致痫成功率为94.74%,模型成功率为73.69%。2、慢性癫痫治疗组在给予LEV治疗2w起,自发性发作次数减少,每日减少至0～2次不等,但未完全缓解;正常对照组无癫痫发作。3、海马重量:癫痫观察组脑重下降15.3%,（癫痫观察组0.137±0.018;正常对照组0.158±0.015,P＜0.01）;慢性癫痫治疗组与慢性癫痫组相比,升高8.1%（EP+LEV组0.149±0.013,EP组0.137±0.018,P＜0.05）;生理盐水对照治疗组较生理盐水对照组下降3.3%（control+LEV组0.153±0.017;control组0.158±0.015,P＞0.05）。慢性癫痫观察组和慢性癫痫观察治疗组mdr1a和mdr1b mRNA表达明显增高于生理盐水对照组和生理盐水对照治疗组,差异有显著统计学意义（P＜0.01）;慢性癫痫观察组mdr1a和mdr1b mRNA表达高于慢性癫痫观察治疗组,差异有统计学意义（P＜0.05）;生理盐水对照治疗组与生理盐水对照组相比,mdr1a和mdr1b mRNA表达有降低的趋势,但无统计学意义（P＞0.05）。4、临床疗效观察:添加治疗7～8w后,安慰剂组28例,2例发作减轻,占7.14%;氟桂利嗪治疗组36例,治疗有效20例（55.56%）,表现为发作次数明显减少,发作持续时间较前减短,发作的严重程度减轻,多次脑电图检测显示癫痫样放电明显减少,与安慰剂组比较差异有显著统计学意义（P＜0.001）。添加治疗前,难治性癫痫组（64例）外周血MDR1mRNA的表达水平明显高于正常对照组（22例）,差异有显著统计学意义（P＜0.01）。添加治疗后,氟桂利嗪治疗组（36例）MDR1 mRNA的表达低于安慰剂组（28例）,但高于正常对照组,差异有统计学意义（P＜0.05,P＜0.01）,安慰剂组MDR1 mRNA的表达比正常对照组高1.14倍,差异有显著统计学意义（P＜0.01）。安慰剂组MDR1 mRNA的表达高于添加治疗前难治性癫痫组,差异有统计学意义（P＜0.05）,氟桂利嗪治疗组MDR1 mRNA的表达低于添加治疗前难治性癫痫组,差异有统计学意义（P＜0.01）。应用氟桂利嗪治疗的患儿2例出现嗜睡,1例出现头晕,不良反应发生率为8.33%。结论1、反复癫痫发作可使Wistar大鼠海马中mdr1a和mdr1b mRNA表达增高。2、抗癫痫新药左乙拉西坦可使大鼠海马多药耐药基因的表达减少。3、左乙拉西坦可使癫痫大鼠海马的重量增加,但使正常大鼠海马重量减轻。4、难治性癫痫患儿MDR1 mRNA在外周血和脑组织同步表达,因此,检测外周血MDR1 mRNA的表达对于临床诊治有重要指导意义。5、氟桂利嗪对MDR1mRNA的表达有逆转作用。可用于小儿难治性癫痫的辅助治疗,能减少癫痫的发作,且小剂量氟桂利嗪副作用小,耐受性好。更多还原

【Abstract】 Background One-third of patients with epilepsy suffer from intractable seizures, which cannot be properly controlled with the current pharmacotherapy.Levetiractam is a new and effective antiepileptic drug,and there is little knowledge about it’s effect on the expression of mdr.So we have make a rat model of spontaneous seizures using kainic acid to study influence of levetiractam to mdr.On the other hand,Flunarizine is a kind of Ca²⁺antagon and some studies show that it can reverse the expression of mdr in rats.But if it can use in the treatment of infractory epilepsy in pediatrics and what is the mechanism of action is still not very clear.Objective We investigated express of mdr1a and mdr1b mRNA and studied whether levetiractam（LEV）affects the expression of mdr1a and mdr1b in the hippocampus of spontaneous seizures rat.To explore the effect and tolerance of flunarizine,which used as an add-on treatment for drug-resistent epilepsy in pediatrics.Methods Spontaneous recurrent seizure were induced by intraperitoneal injection of 1mg/kg kainic acid at postnatal day 7.Control rats were injected with sodium chloride.Then all rats were classified as 4 groups after spontaneous seizures developed: spontaneous seizures（EP,n=13）group,spontaneous seizures treated with LEV （EP+LEV,n=15）group,control（n=16）group and control treated with LEV（control +LEV,n=16）group.The treated rats were given dose of LEV（80mg/kg）,dissolving in 0.9%Sodium Chloride for 10mg/mL,twice a day.All rat were killed at the 56th day of intragastric administration and separate the hippocampus to weigh.The expression of mdr1a and mdr1b mRNA in the hippocampus were measured by RT-PCR.Furthermore,the expression of mdr in peripheral blood of 86 subjects were also tested by PT-PCR.All subjects were divided into intractable epilepsy（64）group and control（22）group,and the intractable epilepsy was made up of flunarizine treatmet （36）and placebo treatment（28）.After 5 mL of the venous blood was collected in each of the patients to detect the expression of MDR1,36 cases received flunarizine 5 mg, po,qn,for 7-8 wk,as an add-on antiepileptic drug.Other 28 cases was given placebo （vitamin B6）10mg,po,qⁿ,for 7-8 wk.All patients were to re-examine the MDR1 mRNA with the same method.Results All rats came to epileptic seizure about 2-10minutes after intraperitoneal injection of KA.At last,the dead number was 8 and there were 2 rats failing.Expression of mdr1a and mdr1b mRNA in the hippocampus were increased significantly in the EP+LEV group and EP group compared with control group （P<0.001）.The EP group was increased compared with EP+LEV group（P<0.05）. control+LEV group have little affect on the expression of mdr1a and mdr1b mRNA in the hippocampus than control group（P >0.05）.Moreover,recurrent seizures causes significant reduction in brain weight:the weight of hippocampus was decreased in mean brain weight of 15.3%（0.137±0.018 vs 0.158±0.015,P<0.01）.LEV led to a increase in weight of brain of 8.1%（0.149±0.013 vs 0.137±0.018）in EP group,but it also resulted in a slight decrease in weight of brain of 3.5%（0.153±0.017 vs 0.158±0.015,P>0.05）in control+LEV group.The expression level of MDR1 mRNA was elevated in the intractable epilepsy, compares with control group（P <0.01）.After treatment with flunarizine about 7-8 wk,20 of 36（55.56%）patients were clinically effective in flunarizine group.At the same time,there was only 2 patient in placebo group（7.14%）effectively.The expression of MDR1 mRNA was decreased in the re-examined 36 cases in flunarizine group,and increased in placebo group,compared with control group.There were 2 cases showing somnolence and 1 case feeling dizziness,the incidence of adverse effect is 8.33%.Conclusion Frequent seizures result in overexpression of mdr1a and mdr1b mRNA in the hippocampus.The expression of mdr1a and mdr1b mRNA in the hippocampus descreased after treatment with levetiractam,so levetiractam could probably lower the level of mdr mRNA.It can promote the development of hippocampus in epileptic rats,but slightly reduces the weight of hippocampus in normal rats.The expression of MDR1 mRNA in peripheral blood is parallel to that in brain,so it can be regarded as an index to evaluate the expression of MDR1 mRNA.Flunarizine is effective,as an add-on therapy in intractable epilepsy with MDR1.It’s mechanism to treat intractable epilepsy may involve in reversing the expression of MDR1 mRNA. At the same time,we find the side effects of flunarizine in low dose are very small,and it can be used as add-on therapy in children intractable epilepsy.更多还原