丙磺舒逆转多药耐药相关蛋白的表达在难治性癫痫治疗中的作用

耐药基因表达成为难治性癫痫重要原因的发现为难治性癫痫的治疗开辟了一条新途径,寻找耐药基因的逆转剂就成为实现这个目标的主要手段。近年来,数10种耐药基因的逆转剂在临床上作过探索,其中最为成熟的药物就是丙磺舒。本文就目前有关丙磺舒在癫痫中的研究进展作一文献复习。 1难治性癫痫中多药耐药相关蛋白的表达及对抗癫痫药物的影响上世纪70年代有学者提出多药耐药的概念。随后在动物和难治性癫痫患者中的研究发现多药耐药基因及其产物P-     

难治性癫痫大鼠脑内多药耐药相关蛋白1表达的研究

目的 研究杏仁核电刺激点燃的难治性癫痫大鼠脑内多药耐药相关蛋白1（multidrug resistant-associated protein 1 MRP1）表达的情况.方法 建立杏仁核电刺激点燃的难治性癫痫大鼠模型,用免疫组织化学及免疫蛋白印记（western blot）的方法,分析比较MRP1蛋白在癫痫模型组与正常对照组的表达.结果 药物难治性癫痫大鼠组脑内多药耐药相关蛋白的表达显著高于正常对照组（P〈0.01）.在癫痫大鼠脑内广泛分布的MRP1免疫阳性细胞主要为毛细血管内皮细胞和星形胶质细胞.结论 癫痫大鼠脑内高表达的MRP1参与了难治性癫痫的耐药机制.     

多药耐药相关蛋白与难治性癫痫

难治性癫痫的发病机制尚不完全清楚,近年来发现在难治性癫痫患者病灶脑组织可检测出多药耐药相关蛋白(MRP)的过度表达,表明MRP与难治性癫痫耐药关系密切。MRP的过度表达受多种因素影响,其可能通过降低抗癫痫药物在血脑屏障的通透性,减少药物的有效浓度,从而引起抗癫痫药物耐药发生。鉴于MRP在难治性癫痫多药耐药中可能的重要作用,筛选非MRP底物的抗癫痫药物和选用适合长期服用的特异性强﹑安全性高﹑毒副作用性小的MRP抑制剂,可能会为今后临床治疗难治性癫痫翻开新的一页。     

药物难治性癫痫患者脑内多药耐药相关蛋白1表达的研究

目的 研究药物难治性癫痫患者脑内皮层多药耐药相关蛋白1（multidrug resistant-associated protein 1，MRP1）表达的情况。方法 选择12例药物难治性癫痫患者癫痫切除灶与12例正常对照脑组织标本．用逆转录聚合酶链反应（RT-PCR）、免疫组化及免疫蛋白印记（Western blot）方法，分析比较MRP1基因在各组的表达。结果 药物难治性癫痫患者组脑内MRP1的表达显著高于正常对照组（P〈0．01）。在癫痫病灶内广泛分布的MRP1免疫阳性细胞主要为毛细血管内皮细胞和星形胶质细胞。结论 脑内高表达的MRP1参与了难治性癫痫的耐药机制。     

多药耐药相关蛋白-1在难治性颞叶癫痫患者脑组织中的表达及丙磺舒的干预作用

目的 观察难治性颞叶癫痫患者脑组织中多药耐药相关蛋白1( MRPl)及应用MRP1拮抗剂丙磺舒干预后的表达,探讨MRP1与难治性颞叶癫痫多药耐药的关系.方法 应用免疫组化检测难治性颞叶癫痫患者脑组织实验组和对照组MRP1的表达情况,同时应用免疫蛋白印记(Western blot)方法检测实验组、丙磺舒干预组和对照组MRP1的表达.结果 免疫组化结果显示MRP1在难治性颞叶癫痫患者脑组织中表达增强,与对照组比较差异有显著性(P＜0.05).Western blot结果显示丙磺舒干预组MRP1蛋白水平较实验组明显较少,差异有显著性(P＜0.05).结论 脑内高表达的MRP1参与难治性颞叶癫痫的耐药机制,丙磺舒可以降低脑组织内MRP1的表达.     

P糖蛋白与难治性癫痫

难治性癫痫(intractable epilepsy)即一般意义上的药物性难治性癫痫(medically intractable epilepsy),在癫痫中约占20％～30％。长期以来,如何阐明难治性癫痫的产生机制,提高对难治性癫痫的控制率一直是癫痫研究的热点。近年,由于受到肿瘤多药耐药(multiple drug resistance,MDR)研究的启示,MDR在难治性癫痫中所扮演的角色逐渐被认识,其中P糖蛋白(P-glycopmtein,PgP)与难治性癫痫的关系研究相对较为深入,本文就此作一综述。 1 Pgp的特性与MDR的关系 Pgp是一分子量为170 kD的膜结合蛋白,转运蛋白ATP结合物(ATP-binding cassette transporters,ABCT)超家族的成员之一,由MDR1基因编码。人类MDR基因分为MDR1和MDR2两种,其中MDR1基因产物Pgp与多药耐药的产生关系非常密切。正常情况下,哺乳动物各种组织均     

多药物耐药蛋白与难治性癫痫关系的研究进展

难治性癫痫患者的多药物耐药已经成为癫痫治疗的焦点问题。本文介绍了目前被认为是主要责任蛋白的P-糖蛋白和多药物耐药相关蛋白的结构与功能,并阐述了其过度表达与癫痫耐药机制的关系及影响其表达的有关因素。对该领域的深入认识将有助于实现难治性癫痫治疗上的突破。     

多药耐药蛋白对大鼠皮层细胞外液卡马西平和苯妥英钠影响的研究

目的观察多药耐药蛋白(multidrugresistance-associatedprotein,MRP)拮抗剂丙磺舒对大鼠大脑皮层细胞外液卡马西平和苯妥英钠含量的影响,证明MRP能够减少皮层内抗癫痫药物的浓度,探讨脑内表达MRP和难治性癫痫多药耐药的关系。方法在健康大鼠大脑皮层内安置微透析探针,腹腔注射卡马西平(20mg/kg)和苯妥英钠(50mg/kg),在给药后不同时间点收集透析液,并用高效液相检测其中的药物浓度,通过微透析探针局部给于丙磺舒,观察后者能否提高大鼠大脑皮层细胞外液卡马西平和苯妥英钠的浓度。结果丙磺舒升高了皮层细胞外液中卡马西平和苯妥英钠的药物浓度,前者在给药后45min～120min显著增高(P<0.05),后者在给药后30min～150min显著增高(P<0.05)。结论MRP具有限制卡马西平和苯妥英钠通过血脑屏障的作用,引起抗癫痫药物在大鼠大脑皮层细胞外液中分布减低,MRP表达增加可能参与了难治性癫痫多药耐药机制的形成。     

难治性癫痫患者脑内MDR1的表达

研究发现大约有三分之一的癫痫患者对抗癫痫药物产生耐药反应。其中又有近60%的患者为难治性癫痫。这类药物难治性癫痫患者的耐药可能机制为脑内多药转运体的过高表达从而导致脑实质内药物浓度的下降。多药耐药基因1(multidrug resistance gene1MDR1)其编码蛋白PGP(P-glycoprotein)是脑内重要的多药转运体。本实验通过逆转录聚合酶链反应(RT-PCR),免疫组织化学和免疫蛋白印记的方法,对12例药物难治性癫痫患者脑内癫痫灶与12例对照组标本的MDR1表达进行研究。1资料与方法1.1研究对象12例药物难治性癫痫患者,均经过适当的抗癫痫药物正规…     

癫痫与多药耐药基因

在过去的20年中，尽管不断有新的抗癫痫药(AEDs)投入临床应用，但仍有30％左右的癫痫患者对AEDs不敏感，而成为难治性癫痫。耐人寻味的是，通常如果患者对一种AED耐受，他往往也对其它的AEDs不敏感，即使这些AEDs的作用途径、结构不同。这就使得认为患者对AEDs交叉耐药的原因是由于引起癫痫发作的机制不同的说法令人怀疑，而倾向于认为有一种共同的机制影响了AEDs的作用。因为这种现象与肿瘤化疗、部分感染性疾病以及器官移植中出现的多药耐药(multidrug resistance)现象相似，故而人们猜测引起肿瘤耐药的机制可能在癫痫的耐药中也有类似存在。     

Major vault protein, a marker of drug resistance, is upregulated in refractory epilepsy

PURPOSE: The molecular basis of drug resistance in epilepsy is being explored. Two proteins associated with drug resistance in cancer, P-glycoprotein and multidrug resistance-associated protein 1, are upregulated in human epileptogenic pathologies. Other proteins associated with resistance in cancer include major vault protein (MVP) and breast cancer resistance protein (BCRP). We hypothesized that these proteins would also be upregulated in human epileptogenic pathologies. METHODS: Hippocampal sclerosis (HS), focal cortical dysplasia (FCD), and dysembryoplastic neuroepithelial tumor (DNT) were studied by using immunohistochemistry for MVP and BCRP. Nonepileptogenic control and histologically normal brain adjacent to epileptogenic tissue were used for comparison. RESULTS: MVP and BCRP were expressed ubiquitously in brain capillary endothelium. Ectopic upregulation of MVP was seen in hilar neurons in HS, dysplastic neurons in FCD, and lesional neurons in DNT. Only in HS cases were rare extralesional neurons immunoreactive. Glial upregulation was not seen. There was no qualitative upregulation of BCRP. CONCLUSIONS: These results show that more than one resistance protein may be upregulated in a given epileptogenic pathology and may contribute to drug resistance. Determination of the types, amounts, and distribution of such proteins will be necessary for rational treatment for drug resistance in epilepsy.     

Multidrug resistance-associated protein is involved in the regulation of extracellular levels of phenytoin in the brain

The mechanisms that lead to drug resistance in epilepsy are not known. Recently, overexpression of multidrug transporters, such as multidrug resistance-associated protein (MRP), has been reported in surgically resected epileptogenic human brain tissue and suggested to contribute to the drug resistance of epilepsy. However, it is not known to what extent multidrug transporters such as MRP are involved in transport of antiepileptic drugs. In the present study, we used in vivo microdialysis in rats to study whether the concentration of phenytoin in the extracellular fluid of the cerebral cortex can be enhanced by inhibition of MRP, using the MRP inhibitor probenecid. Local perfusion with probenecid via the microdialysis probe significantly enhanced the extracellular concentration of phenytoin. The data indicate that MRP critically participates in the regulation of extracellular brain concentrations of the major antiepileptic drug phenytoin.     

Overexpression of the human major vault protein in gangliogliomas

PURPOSE: Recent evidence has been obtained that the major vault protein (MVP) may play a role in multidrug resistance (MDR). We investigated the expression and cellular localization of MVP in gangliogliomas (GGs), which are increasingly recognized causes of chronic pharmacoresistant epilepsy. METHODS: Surgical tumor specimens (n = 30), as well as peritumoral and control brain tissues, were examined for the cellular distribution pattern of MVP with immunocytochemistry. Western blot analysis showed a consistent increase in MVP expression in GGs compared with that in control cortex. RESULTS: In normal brain, MVP expression was below detection in glial and neuronal cells, and only low immunoreactivity (IR) levels were detected in blood vessels. MVP expression was observed in the neuronal component of 30 of 30 GGs and in a population of tumor glial cells. In the majority of the tumors, strong MVP IR was found in lesional vessels. Perilesional regions did not show increased staining in vessels or in neuronal and glial cells compared with normal cortex. However, expression of MVP was detected in the hippocampus in cases with dual pathology. CONCLUSIONS: The increased expression of MVP in GGs is another example of an MDR-related protein that is upregulated in patients with refractory epilepsy. Further research is necessary to investigate whether it could play role in the mechanisms underlying drug resistance in chronic human epilepsy.     

颞叶癫癎主穹窿蛋白表达及其作用研究

目的探讨主穹窿蛋白（major vault protein，MVP）在颞叶癫癎大鼠模型脑组织的表达及其与难治性癫癎耐药是否相关。方法用氯化锂-匹鲁卡品制作颞叶癫癎模型，并将它分为药物有效组和耐药组；用免疫组化和Westernblot法检测MVP表达。结果MVP主要表达在海马和皮质区的小胶质细胞，血管内皮细胞，血管周围的星形胶质细胞也有表达，神经元中少见表达；耐药组MVP表达较药物有效组和对照组明显增高，具有统计学意义（P〈0．05）。结论耐药大鼠模型中MVP过量表达，提示MVP可能与难治性癫癎的耐药性有关。     

Multidrug resistance proteins in tuberous sclerosis and refractory epilepsy

Tuberous sclerosis is an autosomal dominant syndrome characterized by seizures that are refractory to medication in severely affected individuals. The mechanism involved in drug resistance in tuberous sclerosis is unknown. The proteins MDR-1 (multidrug resistance) and MRP-1 (multidrug resistance-associated protein-1) are linked to chemotherapy resistance in tumor cells. However, the relationship between refractoriness to antiepileptic drugs and MDR-1 or MRP-1 brain expression has been poorly studied. We have previously described a case of tuberous sclerosis with refractory epilepsy that expressed multidrug resistance gene (MDR-1) in tuber cells from epileptogenic brain lesion. In this retrospective study, we describe the expression of MDR-1 and MRP-1 in the epileptogenic cortical tubers of three pediatric patients with tuberous sclerosis and refractory epilepsy surgically treated. Monoclonal antibodies for MDR-1 and MRP-1 proteins were used for immunohistochemistry. In epileptogenic cortical tuber brain specimens, MDR-1 and MRP-1 proteins were strongly immunoreactive in abnormal balloon cells, dysplastic neurons, astrocytes, microglial cells, and some blood-brain vessels. A more extensive MDR-1 immunoreactivity was observed. These data suggest that refractory epilepsy phenotype in tuberous sclerosis can be associated with the expression of both multidrug resistance MDR-1 and MRP-1 transporters in epileptogenic cortical tubers.     

P-glycoprotein and multidrug resistance-associated protein are involved in the regulation of extracellular levels of the major antiepileptic drug carbamazepine in the brain.

Despite considerable advances in the pharmacotherapy of epilepsy, about 30% of epileptic patients are refractory to antiepileptic drugs (AEDs). In most cases, a patient who is resistant to one major AED is also refractory to other AEDs, although these drugs act by different mechanisms. The mechanisms that lead to drug resistance in epilepsy are not known. Recently, over-expression of multidrug transporters, such as P-glycoprotein (PGP) and multidrug resistance-associated protein (MRP), has been reported in surgically resected epileptogenic human brain tissue and suggested to contribute to the drug resistance of epilepsy. However, it is not known to what extent multidrug transporters such as PGP or MRP are involved in transport of AEDs. In the present study, we used in vivo microdialysis in rats to study whether the concentration of carbamazepine in the extracellular fluid of the cerebral cortex can be enhanced by inhibition of PGP or MRP, using the PGP inhibitor verapamil and the MRP inhibitor probenecid. Local perfusion with verapamil or probenecid via the microdialysis probe increased the extracellular concentration of carbamazepine. The data indicate that both PGP and MRP participate in the regulation of extracellular brain concentrations of the major AED carbamazepine.     

耐药性癫痫患者血清和脑脊液P-糖蛋白的表达水平变化及其临床意义

目的 观察耐药性癫痫患者血清和脑脊液中多药耐药基因-1、P-糖蛋白的表达水平变化及其临床意义。方法 选取2014年1月-2016年4月本院收治的耐药性癫痫患者以及同期在本院接受抗癫痫药物治疗控制良好的癫痫患者各85例,分别作为耐药性癫痫组和治疗有效组。另选取健康人群85例作为健康对照组。采用酶联免疫吸附测定法及荧光定量聚合酶链反应技术检测所有受试人员的外周血清及脑脊液中多药耐药基因-1及P-糖蛋白表达水平,比较不同类型癫痫患者以上两项指标的表达水平变化。结果 与治疗有效组、健康对照组比较,难治性癫痫组患者血清P-糖蛋白及MDR1-mRNA表达水平较其他2组均明显增高(P<0.05,P<0.01); 治疗有效组与健康对照组比较,血清P-糖蛋白及MDR1-mRNA表达水平无明显差异(P>0.05)。与治疗有效组比较,耐药性癫痫组脑脊液P-糖蛋白及MDR1-mRNA表达水平明显增高(P<0.05)。耐药性癫痫组中不同发作类型患者血清MDR1-mRNA、P-糖蛋白表达水平比较,均无明显差异(P均>0.05)。结论 外周血及脑脊液中多药耐药基因-1、P-糖蛋白的表达水平可以作为癫痫耐药的一项重要指标。     

Expression and cellular distribution of multidrug resistance-related proteins in the hippocampus of patients with mesial temporal lobe epilepsy

PURPOSE: This study investigated the cellular distribution of different multidrug resistance (MDR)-related proteins such as P-glycoprotein (P-gp), the multidrug resistance-associated proteins (MRP) 1 and 2, and the major vault protein (MVP) in normal and sclerotic hippocampus of patients with medically refractory mesial temporal lobe epilepsy (MTLE). METHODS: Single- and double-label immunocytochemistry was used on brain sections of control hippocampus and of hippocampus of refractory MTLE patients. RESULTS: In TLE cases with hippocampal sclerosis (HS), all four MDR proteins examined that had low or no expression in control tissue were upregulated, albeit with different cellular distribution patterns. P-gp immunoreactivity (IR) was observed in astrocytes in regions with diffuse reactive gliosis. In 75% of HS cases, strong P-gp IR was detected in blood vessels, with prominent endothelial labeling. Reactive astrocytes displayed low MRP1 IR. However, glial MRP1 expression was noted in glial endfoot processes around blood vessels. Neuronal MRP1 expression was observed in hypertrophic hilar neurons and in a few residual neurons of the CA1 region. Hippocampal MRP2 expression was observed in the large majority of HS cases in blood vessels. Hypertrophic hilar neurons and blood vessels within the sclerotic hippocampus expressed major vault protein (MVP). CONCLUSIONS: These findings indicate that MDR proteins are upregulated in concert in the hippocampus of patients with refractory MTLE, supporting their role in the mechanisms underlying drug resistance. The specific cell-distribution patterns within the sclerotic hippocampus suggest different cellular functions, not necessarily linked only to clinical drug resistance.     

New proteins configure a brain drug resistance map in tuberous sclerosis

Epileptogenic cortical tubers, characterized by dysplastic neurons and balloon cells, is a frequent feature of tuberous sclerosis. In severe tuberous sclerosis-affected individuals, seizures are refractory to medication. Multidrug resistance proteins (multidrug resistance protein-1 [MDR-1] and multidrug resistance-associated protein-1 [MRP-1]) have been found to be highly expressed in epileptogenic cortical tubers. However, two new proteins related to refractoriness in cancer (breast cancer resistance protein and major vault protein) have not been investigated in tuberous sclerosis and refractory epilepsy. On the same brain specimens previously describing the MDR-1 and MRP-1 expression, we investigated retrospectively breast cancer resistance protein and major vault protein by specific monoclonal antibodies and routine immunohistochemistry methods. Breast cancer resistance protein was present in vascular endothelial cells from all the vessels examined in 3 of 3 cases. Major vault protein was detected in only one case, and selectively expressed in several but not all ballooned cells. In epileptogenic cortical tubers, the additional expression of breast cancer resistance protein on vessels and major vault protein in some ballooned cells to the previously demonstrated expression of MDR-1 and MRP-1 (in vessels, astroglia, microglia, neurons, and ballooned cells) configures a brain protein pharmacoresistance map from patients with tuberous sclerosis and refractory epilepsy.