MDR1基因C3435T、T129C多态性与河南汉族儿童难治性癫痫的相关性

A thesis submitted toZhengzhou Universityfor the degree of MasterThe association between two single nucleotide polymorphismsof multidrug resistance gene 1 and refractory epilepsy inChinese Henan Han childrenBy Yan LiSupervisor：ProfGao LiDepartment of PediatricsThe First Affiliated Hospital Of Zhengzhou UniversityMay 2013原创性声明111111114qlllH IIII llllll lille HlY231 31 97本人郑重声明：所呈交的学位论文，是本人在导师的指导下，独立进行研究所取得的成果。除文中已经注明引用的内容外，本论文不包含任何其他个人或集体已经发表或撰写过的科研成果。对本文的研究作出重要贡献的个人和集体，均已在文中以明确方式标明。本声明的法律责任由本人承担。学位论文作者：杏老 同期：伽凡年口了月l甲同学位论文使用授权声明本人在导师指导下完成的论文及相关的职务作品，知识产权归属郑州大学。根据郑州大学有关保留、使用学位论文的规定，同意学校保留或向国家有关部门或机构送交论文的复印件和电子版，允许论文被查阅和借阅；本人授权郑州大学可以将本学位论文的全部或部分编入有关数据库进行检索，可以采用影印、缩印或者其他复制手段保存论文和汇编本学位论文。本人离校后发表、使用学位论文或与该学位论文直接相关的学术论文或成果时，第一署名单位仍然为郑州大学。保密论文在解密后应遵守此规定。学位论文作者： 硷毙 日期：们一年。，月尸日摘 要MDRl基因C3435T、T129C多态性与河南汉族儿童难治性癫痫的相关性研究生：李岩导师：高丽教授郑州大学第一附属医院儿科中国郑州450052摘要背景与目的癫痫是常见的慢性神经系统疾病，约有近60的患者于儿童时期起病，直接影响全球约5千万人的健康。虽然大多数癫痫患者对抗癫痫药物(antiepilepticdrugs，AEDs)反应良好，但仍有近三分之一的患者治疗无效，属难治性癫痫frefractory epilepsy，RE)。尽管近年来有一些新的抗癫痫药物上市，但并未使难治性癫痫患者的发病率降低。反复的癫痫发作导致患者身体上的残疾和社会适应性降低，是一个严重的全球性的公共卫生问题。是什么原因导致难治性癫痫的产生呢?是否因机体对AEDs产生耐药、其机制又是什么呢?为此，医学界进行了不断的研究。目前研究发现，癫痫药物的耐药机制是一种很复杂的现象，可能涉及多种机制，但具体机制目前并不十分清楚。从国内外对抗肿瘤药物耐药机制的各种研究发现，多药耐药现象与多药耐药(multidrug resistance，MDR)基因1及由其编码的P糖蛋t兰t(permeability glycoprotein，P-gp)有关。Pgp是典型的药物转运蛋白，可将进入大脑或神经元内的药物“泵”出，调控细胞内的药物含量。因此，由于各种原因，使Pgp在血脑屏障上过量表达，可能限制AEDs渗透入大脑，进而导致耐药的发生。2003年Siddiqui等首次发现MDRl C3435T位点的单核苷酸多态。l生(Single Nucleotide Polymorphism，SNP)-与抗癫痫药物的耐药有关，1摘要认为SNP使PgP的表达增加、功能增强，从而减少AED在脑内的分稚水平。自此研究首次提出抗癫痫药物的耐药是Eb基因决定的观点后，众多学者对此进行了大量研究，但结果差异较大，一些研究甚至得出相反的结论。考虑到以上研究结果的差异可能与种族地区差异有关，本文对MDRl基因C3435T、T129C两位点的多态性进行分析，探讨其与我国河南汉族儿章难治性癫痫之间的相关性及儿童难治性癫痫耐药机制形成的分子遗传学机制。方法收集2012年01月一08月于河南省人民医院就诊的癫痫患儿及健康体检患儿的外周血样，共260例，包括难治性癫痫患儿60例，疗效良好的癫痫患儿及健康儿章各100例。采用多聚合酶链反应扩增后继以限制性内切酶片段长度多态性分析方法，分别测定难治性癫痫组、疗效良好组及健康对照组的基因型分布和等位基因频率，应用SPSSl70软件进行统计学分析，以P005认为有统计学差异。9里：日木C3435T位点：难治性癫痫组的基因型以TC为主，其分布频率与疗效良好组之间无相关性，但明显高于健康对照组，且差异有统计学意义俨=0031)，但二者等位基因间无统计学差异。T129C位点：疗效良好组与对照组基因型数目相同，三组均未发现CC基因型，难治组TC基因型分布频率、T等位基因频率高于疗效良好组及健康对照组，差异有统计学意义仁0029，0034)。结论MDRl基因C3435T及T129C的单核苷酸多态性可能与河南汉族儿童难治性癫痫存在相关性，并参与了难治性癫痫耐药机制的形成。关键词： MDRl基因C3435T T129C单核苷酸多态性儿童难治性癫痫AbstractThe association between two single nucleotidepolymorphisms of multidrug resistance gene 1and refractory epilepsy in Chinese Henan HanchildrenPostgraduate Yan LiSupervisor：ProfGao LDepartment of PediatricsThe firSt affiliated hospital of Zhengzhou universityZhengzhou China 450052Backgrounds and 0bj ectiveAbstractEpilepsy is the most prevalent chronic neurological disorder,approximately 60of epileptics had their seizure initially from childhoodAlthough a majority ofpatients with epilepsy are responsive to the presently available antiepileptic drugs，nearlv onethird of them continue to exhibit recurrent seizures，which is calledrefractory epilepsyThe introduction of several new AEDs in the recent years has notimproved the outcome of these patientsPatients with drugresistant epilepsies arephysically and socially disabled，which has become a serious global public healthproblemWhat are the causes of the forming to refractory epilepsy?is it because thebodv resistant to AEDs?Then，what is the mechanisms?To this end，the medicalprofession has made unremitting effortsRecent studies showed that resistance to AED is considered as a complexphenomenon that may involve many mechanisms，none of which is well understoodIIIAbstractThrough the research of the mechanisms for resistance anticancer drugs，we foundthat the resistance is associated with the Pglycoprotein which is encoded byMultidrug resistance gene 1PgP is the archetypal example of multidrug transportproteinsWhich can pump drugs that entered into the brain and neurons，and regulationof intracellular drug contentSo，some rensons that made PgP over expression on thebloodbrain barrier may limit AEDs penetration into the brain，which leading to theoccurrence of resistanceIn 2003，Siddiqui et a1firstly reposed the C3435Tpolymorphism in the MDR 1 gene as being associated with resistance to AEDs，theSingle Nucleotide Polymorphism cound increased the expression of PgP andenhanced its function，thereby reduced the level of AED distributed in the brainSince this study thought that drug resistance in epilepsy might be geneticallydetermined for the first timemany scholars have conducted a lot of research of itwhile，the result is quite different，Some studies have even come to the oppositeconclusionsTaking into account the above findings may be related to racialdifferences in regional disparities，this study will analysis the relationship betweentwo single nucleotide polymorphisms of multidrug resistance gene 1 and Henan Hanchildren with refractory epilepsy,and detect their mechanisms of molecular geneticsMethodsDNA samples were obtained from Henan Provincial PeopleS Hospital，A total of260 cases were enrolled，including 60 cases of refractory epilepsy，1 00 cases ofdrugresponsive epilepsy and 100 cases of healthy childrenThe genotypes and allelefrequencies for MDRl polymorphisms were determined by polymerase chainreactionrestriction fragment length polymorphism analysisSPSS 170 software wasapplied for statistical treatmentP005 was considered statistically significantResuitsC3435T：The genotype of refractory epilepsy group was given priority to withTC，the frequency distribution has no correlation between it and drugresponsivegroup，but significantly higher than healthy control group，and the difference wasIVAbstractstatistically significant(P=O03 0，but there was no statistical difference between thetWO allelesT129C：The drugresponsive group and healthy control group have the samenumber of genotypes，the CC genotype was not found in all of the three groupsThedistribution of TC genotype and T allele frequencies of refractory epilepsy groupdiffered significantly from that in drugresponsive group and healthy control group(P=O029，0034)ConclusionC3435T and T129C polymorphisms of the MDRl gene were associated withrefractory epilepsy in Chinese Henan Han children and may play some role in thecause of resistance mechani