NKCC1和KCC2在致痫性局灶性脑皮质发育不良中表达差异性研究
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摘要
目的探讨钠-钾-氯离子共同转运体1(bumetanide-sensitive sodium-potassium-1-chloride transporter,NKCC1)和钾-氯离子共同转运体2(K+-C1-cotransporter 2,KCC2)在致痫性局灶性脑皮质发育不良中表达差异性情况及其意义。方法依据新疆喀什地区第一人民医院及南方医科大学南方医院病理科的术后病理诊断,参考局灶性脑皮质发育不良(focal cortical dysplasia,FCD)Palmini分型标准,将采集实验标本分为FCDⅠa、Ⅰb、Ⅱa和Ⅱb 4组。实验设置实验组和自身对照组,实验组标本取材于手术中皮层脑电监测提示的癫痫责任灶皮质,自身对照组标本取材于皮层脑电监测提示的非癫痫责任病灶皮质,用实时荧光定量核酸扩增(RT-QPCR)和蛋白质印迹(WB)分别在mRNA水平和蛋白水平分析NKCC1和KCC2在FCD不同亚型间的表达及其差异性。结果 49例病例共分为4组,包括FCDⅠa(10例),FCDⅠb(20例),FCDⅡa(9例),FCDⅡb(10例)4组。WB研究中共纳入47例,RT-QPCR研究中49例病例全部纳入。FCDⅠa亚型中NKCC1和NKCC1mRNA表达与自身对照组比较均未见明显变化,而KCC2表达下调,KCC2mRNA表达上调;FCDⅠb亚型中NKCC1和NKCC1mRNA表达与自身对照组比较均未见明显变化,而KCC2和KCC2mRNA表达均下调;FCDⅡa亚型中NKCC1和NKCC1mRNA表达均上调,而KCC2和KCC2mRNA表达均未见明显变化;FCDⅡb亚型中NKCC1和NKCC1mRNA表达均上调,而KCC2表达下调,KCC2mRNA表达上调。结论只有FCDⅡb亚型同时涉及了NKCC1和KCC2 2种蛋白的表达异常,而其他3种亚型中仅单一涉及NKCC1或KCC2的表达异常;在蛋白表达调节层面,转录层面的调节在NKCC1的表达异常中较为重要,而转录层面和转录后层面的调控在KCC2的表达异常中同时发挥了重要作用。
Objective To investigate the difference of NKCC1 and KCC2 expression in focal cerebral cortex dysplasia caused by epilepsy and its significance.Methods Based on FCD Palmini classification criteria,the experimental specimens were divided into FCD Ⅰa,Ⅰb,Ⅱa andⅡb groups according to the postoperative pathological diagnosis of pathology department of Southern Hospital.All cases were randomly divided into two groups:the experimental group and the control group.The experimental group was taken from the cortex of the epilepsy in the operation.The self-control group was taken from the cortical EEG monitoring prompted the non-epileptic focus cortex.RT-QPCR and WB were carried out to explore the expression and difference of NKCC1 and KCC2 between different subtypes of FCD at mRNA and protein level,repectively.Results 49 cases were divided into 4 groups,including FCD Ⅰa 10 cases,FCD Ⅰb 20 cases,FCD Ⅱa 9 cases,FCD Ⅱb 10 cases.WB study included 47 cases,and RT-QPCR 49 cases.Among FCD Ⅰagroup,NKCC1 showed no obvious change both in mRNA and protein level,while KCC was down-regulated in protein and up-regulated in mRNA level.In FCD Ib cases,NKCC1 was as same as FCDⅠa,but both KCC2 protein and mRNA expression was increased dramatically.In FCD Ⅱagroup,either NKCC1 or NKCC1 mRNA expression was increased statistically,while there was no difference in term of KCC2 in two levels.Among FCD IIb subtype,up-regulation was confirmed concerning NKCC1 protein,NKCC1 mRNA and KCC2 mRNA,but KCC2 protein expression was down-regulated.Conclusion This study was based on the FCD Palmini classification,and the differences in the expression of NKCC1 and KCC2 between FCD subtypes were studied at both protein and mRNA levels.We found that only FCD Ⅱb subtypes had both NKCC1 and KCC2 protein expression abnormalities,while the other three subtypes had only a single expression abnormalities of NKCC1 or KCC2;In the regulation of protein expression level,transcriptional regulation is more important in the expression abnormalities of NKCC1,and regulation of transcriptional and post-transcriptional level in the KCC2 expression abnormalities also played an important role at the same time.
Objective To investigate the difference of NKCC1 and KCC2 expression in focal cerebral cortex dysplasia caused by epilepsy and its significance.Methods Based on FCD Palmini classification criteria,the experimental specimens were divided into FCD Ⅰa,Ⅰb,Ⅱa andⅡb groups according to the postoperative pathological diagnosis of pathology department of Southern Hospital.All cases were randomly divided into two groups:the experimental group and the control group.The experimental group was taken from the cortex of the epilepsy in the operation.The self-control group was taken from the cortical EEG monitoring prompted the non-epileptic focus cortex.RT-QPCR and WB were carried out to explore the expression and difference of NKCC1 and KCC2 between different subtypes of FCD at mRNA and protein level,repectively.Results 49 cases were divided into 4 groups,including FCD Ⅰa 10 cases,FCD Ⅰb 20 cases,FCD Ⅱa 9 cases,FCD Ⅱb 10 cases.WB study included 47 cases,and RT-QPCR 49 cases.Among FCD Ⅰagroup,NKCC1 showed no obvious change both in mRNA and protein level,while KCC was down-regulated in protein and up-regulated in mRNA level.In FCD Ib cases,NKCC1 was as same as FCDⅠa,but both KCC2 protein and mRNA expression was increased dramatically.In FCD Ⅱagroup,either NKCC1 or NKCC1 mRNA expression was increased statistically,while there was no difference in term of KCC2 in two levels.Among FCD IIb subtype,up-regulation was confirmed concerning NKCC1 protein,NKCC1 mRNA and KCC2 mRNA,but KCC2 protein expression was down-regulated.Conclusion This study was based on the FCD Palmini classification,and the differences in the expression of NKCC1 and KCC2 between FCD subtypes were studied at both protein and mRNA levels.We found that only FCD Ⅱb subtypes had both NKCC1 and KCC2 protein expression abnormalities,while the other three subtypes had only a single expression abnormalities of NKCC1 or KCC2;In the regulation of protein expression level,transcriptional regulation is more important in the expression abnormalities of NKCC1,and regulation of transcriptional and post-transcriptional level in the KCC2 expression abnormalities also played an important role at the same time.
引文
[1]吴政.氯离子共转运体KCC2在癫痫发病机制中的作用及其作为抗癫痫靶点的研究[D].复旦大学,2011.
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[8]ADRGNA N C,DI FULVIO M,LAUF P K.Regulation of KCl cotransport:from function to genes[J].J Membr Biol,2004,201(3):109-137.
[9]FLATMAN P W.Regulation of Na-K-2Cl cotransport by phosphorylation andprotein-protein interactions[J].Biochim Biophys Acta,2002,1566(1-2):140-151.
[10]KAHLE K T,STALEY K J,NAHED B V,et al.Roles of the cation-chloridecotransporters in neurological disease[J].Nat Clin Pract Neurol,2008,4(9):490-503.
[11]TALOS D M,SUN H,KOSARAS B,et al.Altered inhibition in tuberous sclerosis andtype IIb cortical dysplasia[J].Ann Neurol,2012,71(4):539-551.
[12]ARONICA E,BOER K,REDEKER S,et al.Differential expression patterns of chloridetransporters,Na+-K+-2Cl-cotransporter and K+-Cl-cotransporter,in epilepsy-associated malformations of cortical development[J].Neuroscience,2007,145(1):185-196.
[13]SHIMIZU-OKABE C,TANAKA M,MATSUDA K,et al.KCC2was downregulated insmall neurons localized in epileptogenic human focal cortical dysplasia[J].Epilepsy Res,2011,93(2/3):177-184.
[14]ROBINSON S,MIKOLAENKO I,THOMPSON I,et al.Loss of cation-chloridecotransporter expression in preterm infants with white matter lesions:implications for the pathogenesis of epilepsy[J].J Neuropathol Exp Neurol,2010,69(6):565-572.
[15]JANSEN L A,PEUGH L D,RODEN W H,et al.Impaired maturation of cortical GABA(A)receptor expression in pediatric epilepsy[J].Epilepsia,2010,51(8):1456-1467.
[16]WANG C,SHIMIZU-OKABE C,WATANABE K,et al.Developmental changes in KCC1,KCC2,and NKCC1 mRNA expressions in the rat brain[J].Brain Res Dev Brain Res,2002,139(1):59-66.
[17]BEN-ARI Y,KHALILOY I,KAHLE KT,et al.The GABA excitatory/inhibitory shift inbrain maturation and neurological disorders[J].Neuroscientist,2012,18(5):467-486.
[18]CONTI L,PALMA E,ROSETI C,et al.Anomalous levels of Cl-transporters cause adecrease of GABAergic inhibition in human peritumoral epileptic cortex[J].Epilepsia,2011,2(9):1635-1644.
[19]TAYLOR D C,FALCONER M A,BRUTON C J,et al.Focal dysplasia of the cerebral cortexin epilepsy[J].J Neurol Neurosurg Psychiatry,1971,34(4):369-387.
[20]SHIMIZU-OKABE C,OKABE A,KILB W,et al.Changes in the expression ofcation-Cl-cotransporters,NKCC1 and KCC2,during cortical malformationinduced by neonatal freeze-lesion[J].Neurosci Res,2007,59(3):288-295.
[3]张慈柳,彭镜,何芳,等.CIC-3氯离子通道蛋白在内侧颞叶癫痫中的表达变化[C]//中华医学会第十七次全国儿科学术大会论文汇编(下册).2012.
[3]GE S,GOH E L,SAILOR K A,et al.GABA regulates synaptic integration of newlygenerated neurons in the adult brain[J].Nature,2006,439(7076):589-593.
[4]CANCEDDA L,FIUMELLI H,CHEN K,et al.Excitatory GABA action is essential formorphological maturation of cortical neurons in vivo[J].J Neurosci,2007,27(19):5224-5235.
[5]REYNOLDS A,BRUSTEIN E,LIAO M,et al.Neurogenic role of the depolarizingchloride gradient revealed by global overexpression of KCC2from the onset ofdevelopment[J].J Neurosci,2008,28(7):1588-1597.
[6]LI H,KHIRUG S,CAI C,et al.KCC2interacts with the dendritic cytoskeleton topromote spine development[J].Neuron,2007,56(6):1019-1033.
[7]PALMINI A,NAJM I,AVANZINI G,et al.Terminology and classification of thecortical dysplasias[J].Neurology,2004,62(6Suppl 3):S2-S8.
[8]ADRGNA N C,DI FULVIO M,LAUF P K.Regulation of KCl cotransport:from function to genes[J].J Membr Biol,2004,201(3):109-137.
[9]FLATMAN P W.Regulation of Na-K-2Cl cotransport by phosphorylation andprotein-protein interactions[J].Biochim Biophys Acta,2002,1566(1-2):140-151.
[10]KAHLE K T,STALEY K J,NAHED B V,et al.Roles of the cation-chloridecotransporters in neurological disease[J].Nat Clin Pract Neurol,2008,4(9):490-503.
[11]TALOS D M,SUN H,KOSARAS B,et al.Altered inhibition in tuberous sclerosis andtype IIb cortical dysplasia[J].Ann Neurol,2012,71(4):539-551.
[12]ARONICA E,BOER K,REDEKER S,et al.Differential expression patterns of chloridetransporters,Na+-K+-2Cl-cotransporter and K+-Cl-cotransporter,in epilepsy-associated malformations of cortical development[J].Neuroscience,2007,145(1):185-196.
[13]SHIMIZU-OKABE C,TANAKA M,MATSUDA K,et al.KCC2was downregulated insmall neurons localized in epileptogenic human focal cortical dysplasia[J].Epilepsy Res,2011,93(2/3):177-184.
[14]ROBINSON S,MIKOLAENKO I,THOMPSON I,et al.Loss of cation-chloridecotransporter expression in preterm infants with white matter lesions:implications for the pathogenesis of epilepsy[J].J Neuropathol Exp Neurol,2010,69(6):565-572.
[15]JANSEN L A,PEUGH L D,RODEN W H,et al.Impaired maturation of cortical GABA(A)receptor expression in pediatric epilepsy[J].Epilepsia,2010,51(8):1456-1467.
[16]WANG C,SHIMIZU-OKABE C,WATANABE K,et al.Developmental changes in KCC1,KCC2,and NKCC1 mRNA expressions in the rat brain[J].Brain Res Dev Brain Res,2002,139(1):59-66.
[17]BEN-ARI Y,KHALILOY I,KAHLE KT,et al.The GABA excitatory/inhibitory shift inbrain maturation and neurological disorders[J].Neuroscientist,2012,18(5):467-486.
[18]CONTI L,PALMA E,ROSETI C,et al.Anomalous levels of Cl-transporters cause adecrease of GABAergic inhibition in human peritumoral epileptic cortex[J].Epilepsia,2011,2(9):1635-1644.
[19]TAYLOR D C,FALCONER M A,BRUTON C J,et al.Focal dysplasia of the cerebral cortexin epilepsy[J].J Neurol Neurosurg Psychiatry,1971,34(4):369-387.
[20]SHIMIZU-OKABE C,OKABE A,KILB W,et al.Changes in the expression ofcation-Cl-cotransporters,NKCC1 and KCC2,during cortical malformationinduced by neonatal freeze-lesion[J].Neurosci Res,2007,59(3):288-295.