儿童癫痫与多药耐药基因表达产物P170关系的初步研究
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摘要
目的：癫痫是神经系统常见疾病之一，其总体预后较好，但仍有相当部分病人用目前的治疗方法难以奏效而成为难治性癫痫，难治性癫痫的发病机制则成为近年来研究的重点，其中耐药基因的表达增高引起了医学界的关注，本实验通过检测多药耐药基因表达产物细胞膜糖蛋白P170在癫痫儿童外周血淋巴细胞的表达水平，初步探讨儿童癫痫与多药耐药基因的相互关系，并指导临床治疗。方法：采用流式细胞仪分析法检测吉林大学第一医院儿科门诊及住院部2003年4月-2003年12月收治的25例癫痫儿童外周血淋巴细胞P170表达水平，均符合目前癫痫的诊断依据：⑴有典型的临床发作；⑵脑电图检查有典型癫痫波；⑶除外非癫痫性发作及其它发作性疾病。其中难治性癫痫组患儿符合目前国内对难治性癫痫的论述：临床经过迁延，频繁的癫痫发作至少每个月4次以上，应用适当的第一线抗癫痫药物正规治疗，药物的血中浓度在有效范围内，无严重的不良反应，至少观察2年仍不能控制发作，影响日常生活同时并无进行性中枢神经系统疾病或占位性病变，排除由于诊断错误、选药不当、用量不足、依从性差等因素所造成的所谓“医源性”难治性癫痫。根据发作类型、脑电图结果、临床治疗效果分为难治性癫痫组、部分性发作癫痫组、全身性发作癫痫组；正常对照组为年龄、性别与研究组无明显差异的健康儿童。结果：（1）各种发作类型癫痫患儿外
周血淋巴细胞P170表达水平均高于正常对照组，经SLD-t检验差异显著（P<0.05），其中难治性癫痫组患儿外周血淋巴细胞P170表达水平(36.1713±5.67%)明显高于正常对照组(1.4980±0.48%)，经SLD-t检验差异显著（P<0.001）；（2）难治性癫痫组患儿与另两组非难治性癫痫组患儿比较，外周血淋巴细胞P170表达水平存在差异，经SLD-t检验有统计学意义（P<0.05）;（3）全身发作性癫痫组与部分发作性癫痫组外周血淋巴细胞P170表达水平(21.2113±4.32%,16.4743±2.04%)相近（P>0.05），无显著差异。结论：P170表达增高可能参与难治性癫痫耐药性的形成；P170的增高可能与癫痫发作及抗癫痫药物诱导有一定的关系，故早期、合理用药，积极控制癫痫的发作可能会减少耐药性形成；P170可以作为难治性癫痫早期诊断的外周指标之一；定期复查P170的表达水平，可以发现耐药性的形成，指导临床合理用药。
Objective: Epilepsy(EP) is a common disease in nervous system, which collective prognosis is preferable, but sill a part of its can’t cure with therapeutic Method at present，which is intractable epilepsy. the pathogenesis of IP becomes the study keystone, and the heighten of MDR’s expression causes medical attention. the objection is studying the relation between the epilepsy in children and multi-drug resistance gene and discussing the likely pathogenesis. Method: To detect the expressive levels of perihemo-lymphcell membrance glyco-protein in 25 children with of epilepsy and 8 healthy children by flow cytometer analytic method. All the studied objects’conditions are coincidence with the diagnosis standards about epilepsy and intractable epilepsy at present, eliminating the other out-breaking diseases. According to the clinical situation.、electroencephalogram results and clinical therapy results, these studied objects are divided into intractable epilepsy group、partial seizure group、total seizure group；the control group is 8 healthy children without difference on age and sex. Results: (1) the expressive levels of perihemo-lymphcell membrance glyco-protein in all 25 children with epilepsy were higher compared to the control group(1.4980±0.48),and the levels of perihemo-lymphcell membrance glyco-protein in children with intractable epilepsy(36.1713±5.67%) were highest ,detected by SLD-t,(P<0.001)there was statistic significance.(2) The expressive levels of perihemo-lymphcell membrance glyco-protein in 8 children
with intractable epilepsy were higher compared to the other children with tractable epilepsy(21.2113±4.32%,16.4743±2.04%) ,detected by SLD-t(P<0.05),there was statistic difference.(3)There were no statistic difference between the two group children with common epilepsy, detected by SLD-t(P>0.05), there was no statistic significance .Conclusion: the heighten expressive level of P170 is likely one of the pathogenesis of intractable epilepsy;P170 may act the diagnostic marker of intractable epilepsy; the expressive levels’ heighten of perihemo-lymphcell membrance in children with epilepsy may caused by diseases itself and ADES itself, So early 、reasonably、 actively control the attack of epilepsy may less the formation of intractable epilepsy; The level of P170 may be helpful to detect the formation of drug-resistance early. Thus the level of P170 in blood has an important clinical significance.
周血淋巴细胞P170表达水平均高于正常对照组，经SLD-t检验差异显著（P<0.05），其中难治性癫痫组患儿外周血淋巴细胞P170表达水平(36.1713±5.67%)明显高于正常对照组(1.4980±0.48%)，经SLD-t检验差异显著（P<0.001）；（2）难治性癫痫组患儿与另两组非难治性癫痫组患儿比较，外周血淋巴细胞P170表达水平存在差异，经SLD-t检验有统计学意义（P<0.05）;（3）全身发作性癫痫组与部分发作性癫痫组外周血淋巴细胞P170表达水平(21.2113±4.32%,16.4743±2.04%)相近（P>0.05），无显著差异。结论：P170表达增高可能参与难治性癫痫耐药性的形成；P170的增高可能与癫痫发作及抗癫痫药物诱导有一定的关系，故早期、合理用药，积极控制癫痫的发作可能会减少耐药性形成；P170可以作为难治性癫痫早期诊断的外周指标之一；定期复查P170的表达水平，可以发现耐药性的形成，指导临床合理用药。
Objective: Epilepsy(EP) is a common disease in nervous system, which collective prognosis is preferable, but sill a part of its can’t cure with therapeutic Method at present，which is intractable epilepsy. the pathogenesis of IP becomes the study keystone, and the heighten of MDR’s expression causes medical attention. the objection is studying the relation between the epilepsy in children and multi-drug resistance gene and discussing the likely pathogenesis. Method: To detect the expressive levels of perihemo-lymphcell membrance glyco-protein in 25 children with of epilepsy and 8 healthy children by flow cytometer analytic method. All the studied objects’conditions are coincidence with the diagnosis standards about epilepsy and intractable epilepsy at present, eliminating the other out-breaking diseases. According to the clinical situation.、electroencephalogram results and clinical therapy results, these studied objects are divided into intractable epilepsy group、partial seizure group、total seizure group；the control group is 8 healthy children without difference on age and sex. Results: (1) the expressive levels of perihemo-lymphcell membrance glyco-protein in all 25 children with epilepsy were higher compared to the control group(1.4980±0.48),and the levels of perihemo-lymphcell membrance glyco-protein in children with intractable epilepsy(36.1713±5.67%) were highest ,detected by SLD-t,(P<0.001)there was statistic significance.(2) The expressive levels of perihemo-lymphcell membrance glyco-protein in 8 children
with intractable epilepsy were higher compared to the other children with tractable epilepsy(21.2113±4.32%,16.4743±2.04%) ,detected by SLD-t(P<0.05),there was statistic difference.(3)There were no statistic difference between the two group children with common epilepsy, detected by SLD-t(P>0.05), there was no statistic significance .Conclusion: the heighten expressive level of P170 is likely one of the pathogenesis of intractable epilepsy;P170 may act the diagnostic marker of intractable epilepsy; the expressive levels’ heighten of perihemo-lymphcell membrance in children with epilepsy may caused by diseases itself and ADES itself, So early 、reasonably、 actively control the attack of epilepsy may less the formation of intractable epilepsy; The level of P170 may be helpful to detect the formation of drug-resistance early. Thus the level of P170 in blood has an important clinical significance.
引文
1.王慕逖,主编.儿科学[M]，第6版，北京:人民卫生出版社，2001：402-403.
2.Mc Donald JW, Silverstein FS,Johnston MV.Neurotoxicity of N-metlyl-D-aspartate is markedly enhanced in developing rat central nervous system[J].Brain Res,1988,459(1):200.
3.Potschka H, Fedrowitz M, Loscher W. P-Glycoprotein and Multidrug Resistance-associated Protein are involved in the regulation of extracellular levels of the major antiepileptic drug carbamazeoine in zhe brain. Neuroreport 2001;12;3557-3560.
4.裘宇容，王伟.肿瘤细胞多药耐药基因及产物P糖蛋白检测方法的研究进展.国外医学.临床生物化学与检验学分册.1999；20（5）：214-215.
5.娄阁，王凌.多药耐药基因.哈尔滨医科大学学报.1996 vol
30,No.3,p.310-312.
6.王春燕，姜振宇，姚程.急性白血病患者多药耐药基因检测的临床意义.吉林大学学报（医学版）.2003 vol.29,No.4,p.
425-427.
7.GropP,et al Mammalian multidrug resistance gene:complete Cdna sequence indicates strong homology to bacterial
transport proteins Cell,1986;47:371.
8.Chang J C,Douglas Kazumits U,et al.Jounal biological Chemistry,1990;265(1):506.
9.Chen CJ,et al Expression of a multidrug-reistance gene in human tumors and tissues Cell,1986;47:381.
10.Cordon-cardo C,et al Expression of the multi-drug resistane gene product（P-glycoprotein） in human nomal andtumor tisues J-H istochem-cytochem,1990;38(9):1277.
11.Salmom SE,et al Multidrug-resistant mycloma: laboratory and clinical effects of verapamil as a chemosensitizer Blood,1991;18(1):44.
12.肖争，晏勇，王学峰.大鼠耐PHA PB杏仁核点燃模型及多药耐受基因的表达[J].中华神经科杂志，1999，32； 365-368.
13.张鑫,潭启富，王卫平等.药物难治性癫痫患者脑内多药耐药基因表达的初步研究.中华神经外科杂志，1999，15（6）：336.
14.Karoly Tm Mary MV Nanct S,et al.Amer Jpathol,1996;149
(3):385.
15. 吴逊，沈鼎烈.难治性癫痫[J]中华神经杂志，1998；31.
16.吴立文.难治性癫痫的诊断和治疗.中华神经科杂 2002，35（1）：55-56.
17.Cynthia E,Herzog J B,Trepel L,et al.J NatCancer Inst,1992,84(9):711.
18.尹格平，孙晓明，陈诵芬.恶性肿瘤细胞多药耐药基因（MDR1）表达产物P170的流式细胞学的检测[J].中国优生与遗传杂
志.1998；6（3）：18-19.
19.Giano CD, Sevlever G,Lazarowki A,et al.Expression of p-glycoprotein and related proteins in brain of patients with refractory temporal lobe epilepsy. Epilepsia, 1997, 38:S41-S43.
20.Lazarowski A,Sevlever G,Taratuto A,et al,Tuberous sclerosis associated with MDR1 gene expression and drug-resistantepilepsy.PediatrNeurol,1999,21;226-228.
21.王学蜂，肖波，孙红斌.难治性癫痫[M].第一版，上海科学技术出版社，2002，45-46.
22.Tisher DM,Weinberg KI,Hinton DR,et al.MDR1 gene expression in brain of patients with medically intractable epilepsy.Epilepsia,1995,36(1):1.
23.吕洋，晏勇，王学蜂，肖争.抗癫痫药体外诱导鼠形胶质细胞多药耐受基因P糖蛋白的表达.中华神经科杂志，2001，34（1）：36-38.
24.Juliano RL.Acta Biochem Biophys,1996;455:152.
25.Rizzi M, Caccia S, Guiso G,et al.Linibic seizures induce P-glycoprote in rodent brain: functional implications for
phar-macoresistance.J Neurosis,2002,22(14):5833.
26.吴瑾，白玉贤，崔滨滨.初发及复发转移大肠癌患者外周血MDR1基因表达的研究.临床肿瘤学杂志，2002 7（5）：321-323.
27.吕喜英，李彦坤，张彦琴.肺癌患者外周学多药耐药基因表达的检测.承德医学院学报，2003 20（1）：15-17.
28.Nakayama M,Wada M,Harada T,et al.Hypomethylation status of CpG sites at the promoter region and overexpression of the human MDR1gene in acute myeloid leukemias.Blood, 1998, 92: 4296-4307.
29.王献，屈宝华，陈品.癫痫与免疫关系的探索.中风与神经疾病杂志，2001 18（2）：109-110.
30.宋先锋，李英良，王云芹.癫痫患者血清细胞因子水平的测定.免疫学杂志，2000 16（6）：473.
31.Karoly Tm Mary M V,Nanct S,et al.Amer J Pathol,1996;149(3):385.
32.Drach J,Gsur A,Hamilton G,et al.Blood,1996;88(5):1747.
2.Mc Donald JW, Silverstein FS,Johnston MV.Neurotoxicity of N-metlyl-D-aspartate is markedly enhanced in developing rat central nervous system[J].Brain Res,1988,459(1):200.
3.Potschka H, Fedrowitz M, Loscher W. P-Glycoprotein and Multidrug Resistance-associated Protein are involved in the regulation of extracellular levels of the major antiepileptic drug carbamazeoine in zhe brain. Neuroreport 2001;12;3557-3560.
4.裘宇容，王伟.肿瘤细胞多药耐药基因及产物P糖蛋白检测方法的研究进展.国外医学.临床生物化学与检验学分册.1999；20（5）：214-215.
5.娄阁，王凌.多药耐药基因.哈尔滨医科大学学报.1996 vol
30,No.3,p.310-312.
6.王春燕，姜振宇，姚程.急性白血病患者多药耐药基因检测的临床意义.吉林大学学报（医学版）.2003 vol.29,No.4,p.
425-427.
7.GropP,et al Mammalian multidrug resistance gene:complete Cdna sequence indicates strong homology to bacterial
transport proteins Cell,1986;47:371.
8.Chang J C,Douglas Kazumits U,et al.Jounal biological Chemistry,1990;265(1):506.
9.Chen CJ,et al Expression of a multidrug-reistance gene in human tumors and tissues Cell,1986;47:381.
10.Cordon-cardo C,et al Expression of the multi-drug resistane gene product（P-glycoprotein） in human nomal andtumor tisues J-H istochem-cytochem,1990;38(9):1277.
11.Salmom SE,et al Multidrug-resistant mycloma: laboratory and clinical effects of verapamil as a chemosensitizer Blood,1991;18(1):44.
12.肖争，晏勇，王学峰.大鼠耐PHA PB杏仁核点燃模型及多药耐受基因的表达[J].中华神经科杂志，1999，32； 365-368.
13.张鑫,潭启富，王卫平等.药物难治性癫痫患者脑内多药耐药基因表达的初步研究.中华神经外科杂志，1999，15（6）：336.
14.Karoly Tm Mary MV Nanct S,et al.Amer Jpathol,1996;149
(3):385.
15. 吴逊，沈鼎烈.难治性癫痫[J]中华神经杂志，1998；31.
16.吴立文.难治性癫痫的诊断和治疗.中华神经科杂 2002，35（1）：55-56.
17.Cynthia E,Herzog J B,Trepel L,et al.J NatCancer Inst,1992,84(9):711.
18.尹格平，孙晓明，陈诵芬.恶性肿瘤细胞多药耐药基因（MDR1）表达产物P170的流式细胞学的检测[J].中国优生与遗传杂
志.1998；6（3）：18-19.
19.Giano CD, Sevlever G,Lazarowki A,et al.Expression of p-glycoprotein and related proteins in brain of patients with refractory temporal lobe epilepsy. Epilepsia, 1997, 38:S41-S43.
20.Lazarowski A,Sevlever G,Taratuto A,et al,Tuberous sclerosis associated with MDR1 gene expression and drug-resistantepilepsy.PediatrNeurol,1999,21;226-228.
21.王学蜂，肖波，孙红斌.难治性癫痫[M].第一版，上海科学技术出版社，2002，45-46.
22.Tisher DM,Weinberg KI,Hinton DR,et al.MDR1 gene expression in brain of patients with medically intractable epilepsy.Epilepsia,1995,36(1):1.
23.吕洋，晏勇，王学蜂，肖争.抗癫痫药体外诱导鼠形胶质细胞多药耐受基因P糖蛋白的表达.中华神经科杂志，2001，34（1）：36-38.
24.Juliano RL.Acta Biochem Biophys,1996;455:152.
25.Rizzi M, Caccia S, Guiso G,et al.Linibic seizures induce P-glycoprote in rodent brain: functional implications for
phar-macoresistance.J Neurosis,2002,22(14):5833.
26.吴瑾，白玉贤，崔滨滨.初发及复发转移大肠癌患者外周血MDR1基因表达的研究.临床肿瘤学杂志，2002 7（5）：321-323.
27.吕喜英，李彦坤，张彦琴.肺癌患者外周学多药耐药基因表达的检测.承德医学院学报，2003 20（1）：15-17.
28.Nakayama M,Wada M,Harada T,et al.Hypomethylation status of CpG sites at the promoter region and overexpression of the human MDR1gene in acute myeloid leukemias.Blood, 1998, 92: 4296-4307.
29.王献，屈宝华，陈品.癫痫与免疫关系的探索.中风与神经疾病杂志，2001 18（2）：109-110.
30.宋先锋，李英良，王云芹.癫痫患者血清细胞因子水平的测定.免疫学杂志，2000 16（6）：473.
31.Karoly Tm Mary M V,Nanct S,et al.Amer J Pathol,1996;149(3):385.
32.Drach J,Gsur A,Hamilton G,et al.Blood,1996;88(5):1747.