【作者】 孔敏；

【导师】 蒋莉；

【作者基本信息】 重庆医科大学 ， 儿科学， 2013， 博士

【摘要】 第一部分癫痫患儿外周血多药耐药基因及P-糖蛋白的动态表达及意义研究背景：癫痫在儿童和青少年时期有较高的发病率，对儿童的生长发育、生活和学习造成了很大的影响。癫痫治疗仍以长期口服抗癫痫药物（AEDs）为主，大约30%的患儿治疗无效呈难治性癫痫(RE)。多药耐药是临床治疗的难点，研究发现多药耐药基因1（MDR1）及其表达产物P-糖蛋白（P-gp）的过度表达使AEDs进入脑组织的能力下降，血药浓度降低，进而使药物疗效降低癫痫发作不能有效控制。目前关于MDR1和P-gp在评估儿童AEDs的疗效、预测儿童难治性癫痫的研究较少。目的：研究癫痫患儿外周血多药耐药基因及其表达产物P-糖蛋白表达的动态变化，探讨其与癫痫耐药的关系及预测儿童抗癫痫药物疗效的价值。方法：选取2011年1月至2012年12月重庆医科大学附属儿童医院神经内科门诊和病房就诊的难治性癫痫患儿30例、初诊癫痫患儿40例及健康体检儿童30例，分别于入组前、治疗后6、12和18个月，应用逆转录聚合酶链反应（RT-PCR）半定量法检测外周血MDR1mRNA的表达、流式细胞术检测P-gp的表达，分析MDR1mRNA、P-gp表达及其表达水平与癫痫发作频率和应用AEDs抗癫痫疗效的关系。结果：（1）儿童外周血MDR1mRNA表达水平与P-gp表达高度正相关（r＝0.82，P＜0.05）。（2）难治性癫痫组、初诊癫痫组入组前，治疗后6、12和18个月MDR1mRNA、P-gp表达较对照组均显著增高（P＜0.01）；难治性癫痫组各个观察时间点MDR1mRNA、P-gp表达较初诊癫痫组显著增高（P＜0.01）；难治性癫痫组MDR1mRNA、P-gp表达治疗18个月较入组前明显增高（P＜0.01），治疗12个月较入组前及治疗6个月、治疗18个月较治疗6个月、12个月均增高（P＜0.05）；初诊癫痫组各个观察时间点MDR1mRNA、P-gp的表达比较差异无统计学意义（P＞0.05）。（3）难治性癫痫组入组前MDR1mRNA、P-gp表达阳性19例中16例（84.2%）治疗无效，表达阴性11例中3例（27.3%）无效，两者比较差异有统计学意义（P＜0.01）；初诊癫痫组入组前MDR1mRNA、P-gp表达阳性13例中9例（69.2%）转化为难治性癫痫，表达阴性27例中2例（7.5%）转化为难治性癫痫，两者比较差异有统计学意义（P＜0.01）。（4）发作频繁患儿MDR1mRNA、P-gp水平明显高于发作较少患儿（P＜0.01）。（5）使用4种AEDs患儿MDR1mRNA、P-gp水平明显高于使用2种及3种AEDs（P＜0.01）。结论：（1）癫痫患儿外周血MDR1mRNA、P-gp表达升高，难治性癫痫升高更明显，可能是癫痫临床耐药的重要原因。（2）外周血MDR1mRNA、P-gp可作为评估AEDs疗效的客观指标，可预测难治性癫痫发生的可能。（3）定时定量检测癫痫患儿外周血MDR1mRNA、P-gp表达的动态变化，可评估AEDs的疗效，指导用药，尽量减少难治性癫痫的发生。第二部分常用抗癫痫药对癫痫儿童外周血P-gp表达的影响研究背景：临床多药耐药是癫痫治疗的难点，目前研究认为MDR1基因编码的P-gp的过度表达与癫痫耐药密切相关。其表达增高是否为先天耐药亦或是癫痫发作或AEDs应用诱导的结果，具体原因不清楚，且评估儿童AEDs疗效的客观指标较少。目的：以50例初诊癫痫患儿为研究对象，探讨丙戊酸钠、左乙拉西坦、奥卡西平对初诊癫痫患儿外周血P-gp表达的影响及P-gp在预测儿童难治性癫痫的价值。方法：50例初诊癫痫患儿根据治疗方案分为丙戊酸钠组（n=19）、左乙拉西坦组（n=16）和奥卡西平组（n=15），20例健康体检儿童为对照组。分别于治疗前、治疗后6和12个月应用流式细胞术检测各组儿童外周血P-gp的表达。分析P-gp表达及其表达水平与抗癫痫疗效的关系。结果:（1）丙戊酸钠组、左乙拉西坦组和奥卡西平组治疗前，治疗后6和12个月P-gp表达较对照组均显著增高（P＜0.01）；丙戊酸钠组、左乙拉西坦组、奥卡西平组各个观察时间点P-gp水平组内及组间比较差异均无统计学意义（P均＞0.05）。（2）治疗无效患儿外周血P-gp表达明显高于治疗有效和对照组患儿（P均＜0.01）；治疗有效患儿和对照组比较差异无统计学意义。（3）50例患儿治疗前P-gp表达阳性15例中9例（69.2%）转化为难治性癫痫，表达阴性35例中3例（7.5%）转化为难治性癫痫，两者比较差异有统计学意义（P＜0.01）。结论：（1）癫痫患儿外周血P-gp表达升高，难治性癫痫升高更明显，可能是癫痫临床耐药的重要原因。（2）外周血P-gp可作为评估AEDs疗效的客观指标，可预测难治性癫痫发生的可能。（3）丙戊酸钠、左乙拉西坦和奥卡西平单药治疗不影响癫痫患儿外周血P-gp的表达，其表达增高可能是反复痫性发作或先天耐药所致。第三部分常用抗癫痫药对大鼠体内多药耐药基因及P-糖蛋白表达的影响研究背景：多药耐药基因1（MDR1）及其编码产物P-糖蛋白（P-gp）在癫痫病灶的异常高表达被认为是癫痫耐药的重要机制，但其表达增高是先天遗传获得或是后天因素诱导所致，具体原因并不清楚。多数学者认为反复癫痫发作可诱导MDR1和P-gp表达增高，但对于AEDs是否会导致其表达增高仍存有争议。近年研究发现癫痫患者除病变脑组织MDR1和P-gp表达增高外，其外周血表达亦明显活跃，但二者是否具有相关性，外周血的表达能否代替脑组织，目前研究较少。目的：以氯化锂-匹罗卡品诱发的慢性自发性癫痫大鼠及正常大鼠为研究对象，建立长期抗癫痫药物治疗动物模型，探讨常用抗癫痫药物（AEDs）丙戊酸钠（VPA）、奥卡西平（OXC）、左乙拉西坦（LEV）对大鼠外周血多药耐药基因1（MDR1）及脑组织P-糖蛋白（P-gp）表达的影响。方法：随机将75只雄性4-5周SD大鼠分为氯化锂-匹罗卡品诱发慢性癫痫模型组（43只）和对照组（32只），将造模成功后存活的32只大鼠根据使用AEDs的不同，随机分为VPA治疗（EP+VPA）组、OXC治疗（EP+OXC）组、LEV治疗（EP+LEV）及癫痫未治疗（EP）组，每组动物8只。对照组随机分为正常对照（NS）组、VPA治疗（NS+VPA）组、OXC治疗（NS+OXC）组及LEV治疗（NS+LEV）组，每组动物8只。灌胃给药4周，采用逆转录聚合酶链反应（RT-PCR）半定量法检测外周血MDR1mRNA的表达、免疫组化Envision法检测脑组织P-gp的表达。结果：（1）大鼠外周血MDR1mRNA表达水平与脑组织P-gp表达高度正相关（r＝0.71，P＜0.05）。（2）与正常对照组比较，EP组、EP+VPA组、EP+OXC组及EP+LEV组各组大鼠外周血MDR1mRNA、脑组织P-gp表达均显著增高（P＜0.01）；NS+VPA组、NS+OXC组及NS+LEV组各组MDR1mRNA、P-gp表达比较差异无统计学意义（P＞0.05）。（3）与EP组比较，EP+VPA组、EP+OXC组及EP+LEV组各组大鼠外周血MDR1mRNA、脑组织P-gp表达水平差异无统计学意义（P＞0.05）。结论：（1）癫痫大鼠外周血MDR1mRNA、脑组织P-gp表达均升高，可能在癫痫耐药的发生机制中发挥了重要作用。（2）癫痫大鼠外周血MDR1mRNA水平可以作为脑组织P-gp表达可靠的简易的参考指标。（3）单纯AEDs VPA、OXC、LEV的使用不影响大鼠外周血MDR1mRNA、脑组织P-gp的表达，癫痫大鼠MDR1mRNA、P-gp的高表达可能是反复癫痫发作未控制的结果。更多还原

【Abstract】 PART ONE THE DYNAMIC EXPRESSION ANDSIGINIFICANCE OF MULTIDRUG RESISTANCE GENEAND P-GLYCOPROTEIN IN THE PERIPHERAL BLOODOF CHILDREN WITH EPILEPSYBackground:Epilepsy has a higher incidence in children and adolescence, which hasa great impact on children ’s growth and development and life andlearning. The main treatment of epilepsy still is long-term oralantiepileptic drugs (AEDs), approximately30%of children withepilepsy become refractory epilepsy(RE).Multidrug resistance is thedifficulty of clinical treatment. Previous studies have shown that theoverexpression of multidrug resistance gene1(MDR1) and its codedproduct P-glycoprotein (P-gp) reduced the ability of AEDs to enterbrain tissue and reduced blood concentration, and thus the seizurescouldn’t be effectively controlled. Currently, the studies on MDR1and P-gp as objective indicators of assessing AEDs efficacy and predictingrefractory epilepsy with children have fewer reported.Objective:To observe dynamic changes of the expression of multidrug resistancegene1and P-glycoprotein in peripheral blood in children withepilepsy，explore the relationship between it and epileptic resistanceand its value of predicting efficacy of AEDs.Methods:A total of70inpatients and outpatients in our department from January2011to December2o12were subjected in this study. These childrenwere divided into refractory epilepsy (n=30), and newly diagnosedepilepsy (n=40). Another30healthy children served as normal control.The expression of MDR1mRNA was analyzed by reverse transcriptionpolymerase chain reaction （RT-PCR） semi-quantitatively and P-gpwas detected by flow cytometry before entering group，6months，12months and18months after treatment， respectively. Therelationship of MDR1mRNA and P-gp level with epileptic frequencyand therapeutic effect of AEDs was analyzed.Results:(1) A positive correlation was found between the expression ofMDR1mRNA and P-gp in peripheral blood in children（r=0.86，P<0.05）. (2) The expression of MDR1mRNA and P-gp in refractory epilepsygroup and newly diagnosed epilepsy group at every time pointwere all significantly higher than that in healthy control group（P＜0.01）. The expression of MDR1mRNA and P-gp refractoryepilepsy group at every time point were all significantly higherthan that in newly diagnosed epilepsy（P＜0.01）. There was asignificant difference of the expression of the MDR1mRNA andP-gp in refractory epilepsy group at every time point（P＜0.01， P＜0.05）and not significantly different in newly diagnosed epilepsygroup.(3) In refractory epilepsy group before entering group，16out of19cases（84.2%）with over expression of MDR1mRNA and P-gpbecame invalid，3out of11cases（27.3%） without expression ofMDR1mRNA and P-gp became invalid，and there was a significantdifference between them（P＜0.01）. In newly diagnosed epilepsygroup before entering group，9out of13cases （69.2%）with overexpression of MDR1mRNA and P-gp became intractable epilepticpatients and2out of27（7.5%）without expression of MDR1mRNAand P-gp became intractable epileptic patients， there was asignificant difference between them（P＜0.01）.(4) MDR1mRNA and P-gp expression was more among patients withhigh frequent epilepsy than patients with low frequent epilepsy（P ＜0.01）. MDR1mRNAand P-gp expression was more in patientsadministered with four kinds of AEDs than those with two or threekinds of AEDs（P＜0.01）.Conclusion:(1) The expression of MDR1mRNA and P-gp increase in peripheralblood of children with epilepsy，especially in refractory epilepticchildren. The increased expression of MDR1mRNA and P-gp mayplay an important role in clinical drug resistance.(2) The expression of MDR1mRNA or P-gp may be a predictablemarker of evaluating efficacy of AEDs and occurring ofrefractory epilepsy.(3) The regular and quantitative detection of dynamic changes of theexpression of MDR1mRNA or P-gp in peripheral blood in childrenwith epilepsy, which can evaluate efficacy of AEDs and guidetreatment and minimize occurrence of refractory epilepsy. PART TWO EFFECT OF ANTIEPILEPTIC DRUGS ONEXPRESSION OF P-GLYCOPROTEIN OF CHILDRENBackground:Multidrug resistance is the difficulty of treatment of epilepsy, previousstudies have shown that the overexpression of P-gp encoded by MDR1had a close relationship with epilepsy drug resistance. But the specificreasons of its overexpression are still unclear, it maybe obtain fromhereditary or from induction of acquired factors including repeatedepileptic seizures or AEDs. Currently, the studies on objectiveindicators of assessing AEDs efficacy and predicting refractoryepilepsy with children have fewer reported.Objective:Fourty children of newly diagnosed epilepsy inpatients and outpatientsin our department were subjected in this study, to explore the effects ofvalproate acid, oxcarbazepine, levetiractam on expression ofP-glycoprotein(P-gp) in peripheral blood and its value of predictingefficacy of AEDs in children.MethodsFourty children of newly diagnosed epilepsy were divided intovalproate acid group(n=19), oxcarbazepine group(n=16), levetiractamgroup(n=15), another20healthy children served as normal control. The expression of P-gp was detected by flow cytometry beforetreatment，6months，12months and18months after treatment，respectively. The relationship of P-gp level with therapeutic effect ofAEDs was analyzed.Results:(1) The expression of P-gp in valproate acid group, oxcarbazepinegroup and levetiractam group at every time point were allsignificantly higher than that in healthy control group（P＜0.01）;there was no significant difference of the expression of P-gp invalproate acid group, oxcarbazepine group and levetiractam groupat every time point.(2) The expression of P-gp was higher of patients with invalidtreatment than that in patients with valid treatment and healthycontrol group（P＜0.01）.(3) In50paitents, before treatment，9out of15cases（69.2%）withover expression of P-gp became invalid，3out of35cases（7.5%）without expression of P-gp became invalid，and there was asignificant difference between them （P＜0.01）.Conclusion:(1) The expression of P-gp increase in peripheral blood of childrenwith epilepsy， especially in refractory epileptic children. Theincreased expression of P-gp may play an important role in clinical drug resistance.(2) The expression of P-gp may be a predictable marker of evaluatingefficacy of AEDs and occurring of refractory epilepsy.(3) There was no influence on the expression of P-gp in peripheralblood of mere use of valproate acid, oxcarbazepine andlevetiractam, the increased expression of P-gp may be theconsequence of uncontrolled recurrent seizures. PART THREE EFFECT OF ANTIEPILEPTIC DRUGS ONEXPRESSION OF MULTIDRUG RESISTANCE GENEAND P-GLYCOPROTEIN OF RATSBackground:The abnormally overexpression of multidrug resistance gene1(MDR1)and its coded product P-glycoprotein (P-gp) in epileptic focus havebeen considered to be an important mechanism of the epilepsy drug.But the specific reasons of their overexpression are still unclear, theymaybe obtain from hereditary or from induction of acquired factors.Many scholars regard that repeated seizures can induce overexpressionof MDR1and P-gp, but there is still controversial for the AEDswhether it will lead to overexpression. Previous studies have shownthat the expression of MDR1and P-gp in peripheral blood are alsoactive except that overexpression in Lesions brain tissue of patientswith epilepsy. But whether there is a relationship between theexpression in peripheral blood and brain tissue, whether the expressionin peripheral blood can instead brain tissue, the related study is stillless.Objective:Chronic and spontaneous epileptic rats induced by lithium-pilocarpineand normal rats were used to be study objects, and animal model of long-term antiepileptic drugs treatment was established to explore theeffects of valproate acid (VPA), oxcarbazepine (OXC), levetiractam(LEV) on expression of multidrug resistance gene1（MDR1）inperipheral blood and P-glycoprotein(P-gp) in brain.Methods:75male rats of4-5weeks were randomly divided into chronic epilepsymodel group （n=43） induced by lithium-pilocarpine and controlgroup(n=32). When epilepsy model was developed，the32survivedrats were divided into VPA group (EP+VPA）, OXC group (EP+OXC）,LEV group (EP+LEV）and EP group. Rats of control group weredivided into normal saline(NS) group, VPA group (NS+VPA）, OXCgroup (NS+OXC）and LEV group (NS+LEV）. Gavage twice daily for4weeks, the expression of MDR1mRNA in peripheral blood wasanalyzed by reverse transcription polymerase chain reaction （RT-PCR） semi-quantitatively and P-gp was detected byimmunohistochemical method.Results:(1) A positive correlation was found between the expression ofMDR1mRNA in peripheral blood and P-gp in brain in rats（r=0.71， P <0.05）.(2) The expression of MDR1mRNA in peripheral blood and P-gp inbrain in EP group, EP+VPA group, EP+OXC group and EP+LEV group were all significantly higher than that in NS group（P＜0.01）.There was not significantly different in NS+VPA group,NS+OXC group and NS+LEV group（P＞0.05）.(3) Compared with EP group, there was not significantly different inEP+VPA group, EP+OXC group and EP+LEV group of theexpression of MDR1mRNA in peripheral blood and P-gp in brain（P＞0.05）.Conclusion:(1) The expression of MDR1mRNA in peripheral blood and P-gp inbrain increase of rats with epilepsy，their overexpression may playan important role in the drug resistance of epilepsy.(2) The expression of MDR1mRNA in peripheral blood can be used asa credible and simple indicator of the expression of P-gp in braintissue of epileptic rats.(3) There was no influence on the expression of MDR1mRNA inperipheral blood and P-gp in brain of mere use of VPA or OXC orLEV, the increased expression of MDR1mRNA and P-gp may bethe consequence of uncontrolled recurrent seizures.更多还原