难治性颞叶内侧型癫痫患者基因差异表达探讨
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摘要
目的应用基因芯片技术检测难治性颞叶内侧型癫痫患者的硬化海马组织与正常海马组织基因差异表达,从基因水平探讨颞叶癫痫的发病机制。方法收集2015年4月-2017年6月于解放军总医院第一医学中心手术切除获得的3例难治性颞叶内侧型癫痫患者硬化海马组织与尸检获得的3例正常海马组织,分别提取样本中总RNA,逆转录合成cDNA,荧光分子标记后,利用芯片进行检测,获取差异表达基因。采用qRT-PCR实验对差异表达基因进行验证。利用生物信息学软件进一步对差异表达基因进行GO功能与Passway通路分析。结果与正常海马组织相比,硬化海马组织共有399个mRNAs有差异性表达(差异倍数> 2.0或<-2.0,P <0.05),其中mRNA高表达的有236个,mRNA低表达的有163个,基因差异表达最高与最低的为CPLX3与DCDC5。差异基因涉及的功能与通路有炎症反应、信号受体活性、神经肽受体活性及信号通路、神经活性配体-受体相互作用通路、钙离子信号通路。结论难治性颞叶内侧型癫痫患者硬化海马组织与正常海马组织有明显差异表达基因,对差异基因功能与通路的研究进一步揭示了颞叶内侧型癫痫的发病机制。
Objective To detect the differential gene expression of sclerosis hippocampus in patients with refractory mesial temporal lobe epilepsy(MTLE) and normal hippocampus by gene chip technique, and explore the pathogenesis of temporal lobe epilepsy from the genetic aspect. Methods From April 2015 to June 2017, three sclerosis hippocampus obtained from patients with MTLE who underwent refractory temporal lobe epilepsy surgery and three normal hippocampus obtained from autopsy were collected in our study. Total RNA was extracted from the samples, and cDNA was synthesized by reverse transcription. After fluorescent labeling, the chip was used to detect differentially expressed genes. The differentially expressed genes were verified by qRT-PCR assay. GO function and Pathway analysis were further performed based on the differential expression of mRNAs using bioinformatic software. Results Compared with normal hippocampus, 399 mRNAs differentially expressed in sclerosis hippocampus, consisting of 236 upregulated and 163 downregulated mRNAs(fold-change>2.0 or<-2.0, P <0.05). The highest and lowest differential expressing genes were CPLX3 and DCDC5, respectively. The functions and pathways involved in differential genes included inflammatory response, signal receptor activity, neuropeptide receptor activity and signaling pathway, neuroactive ligand-receptor interaction pathway, and calcium signaling pathway. Conclusion Compared with normal hippocampus, the genes significantly and differentially express in sclerosis hippocampus in patients with MLTE. Go function and pathway analyses of differential expression genes can reveal the pathogenesis of MTLE.
Objective To detect the differential gene expression of sclerosis hippocampus in patients with refractory mesial temporal lobe epilepsy(MTLE) and normal hippocampus by gene chip technique, and explore the pathogenesis of temporal lobe epilepsy from the genetic aspect. Methods From April 2015 to June 2017, three sclerosis hippocampus obtained from patients with MTLE who underwent refractory temporal lobe epilepsy surgery and three normal hippocampus obtained from autopsy were collected in our study. Total RNA was extracted from the samples, and cDNA was synthesized by reverse transcription. After fluorescent labeling, the chip was used to detect differentially expressed genes. The differentially expressed genes were verified by qRT-PCR assay. GO function and Pathway analysis were further performed based on the differential expression of mRNAs using bioinformatic software. Results Compared with normal hippocampus, 399 mRNAs differentially expressed in sclerosis hippocampus, consisting of 236 upregulated and 163 downregulated mRNAs(fold-change>2.0 or<-2.0, P <0.05). The highest and lowest differential expressing genes were CPLX3 and DCDC5, respectively. The functions and pathways involved in differential genes included inflammatory response, signal receptor activity, neuropeptide receptor activity and signaling pathway, neuroactive ligand-receptor interaction pathway, and calcium signaling pathway. Conclusion Compared with normal hippocampus, the genes significantly and differentially express in sclerosis hippocampus in patients with MLTE. Go function and pathway analyses of differential expression genes can reveal the pathogenesis of MTLE.
引文
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5韩春雷,葛燕,孟凡刚,等.海人酸致颞叶癫痫大鼠基因差异表达的研究[J].中华实验外科杂志,2014,31(3):567-569.
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7 Bungenberg J,Surano N,Grote A,et al.Gene expression variance in hippocampal tissue of temporal lobe epilepsy patients corresponds to differential memory performance[J].Neurobiol Dis,2016,86:121-130.
8 Zubareva OE,Kovalenko AA,Kalemenev SV,et al.Alterations in mRNA expression of glutamate receptor subunits and excitatory amino acid transporters following pilocarpine-induced seizures in rats[J].Neurosci Lett,2018,686:94-100.
9 Mohrmann R,Dhara M,Bruns D.Complexins:small but capable[J].Cell Mol Life Sci,2015,72(22):4221-4235.
10 Chang S,Reim K,Pedersen M,et al.Complexin stabilizes newly primed synaptic vesicles and prevents their premature fusion at the mouse calyx of held synapse[J].J Neurosci,2015,35(21):8272-8290.
11 Lin MY,Rohan JG,Cai H,et al.Complexin facilitates exocytosis and synchronizes vesicle release in two secretory model systems[J].J Physiol,2013,591(10):2463-2473.
12 Landgraf I,Mühlhans J,Dedek K,et al.The absence of Complexin 3 and Complexin 4 differentially impacts the ON and OFF pathways in mouse retina[J].Eur J Neurosci,2012,36(4):2470-2481.
13 Mortensen LS,Park SJH,Ke JB,et al.Complexin 3 Increases the Fidelity of Signaling in a Retinal Circuit by Regulating Exocytosis at Ribbon Synapses[J].Cell Rep,2016,15(10):2239-2250.
14 Kurokawa A,Narukawa M,Ohmoto M,et al.Expression of the synaptic exocytosis-regulating molecule complexin 2 in taste buds and its participation in peripheral taste transduction[J].J Neurochem,2015,133(6):806-814.
15 Babai N,Sendelbeck A,Regus-Leidig H,et al.Functional Roles of Complexin 3 and Complexin 4 at Mouse Photoreceptor Ribbon Synapses[J].J Neurosci,2016,36(25):6651-6667.
16 Xue M,Stradomska A,Chen H,et al.Complexins facilitate neurotransmitter release at excitatory and inhibitory synapses in mammalian central nervous system[J].Proc Natl Acad Sci USA,2008,105(22):7875-7880.
17 Quintana-Pájaro LJ,Ramos-Villegas Y,Cortecero-Sabalza E,et al.The Effect of Statins in Epilepsy:A Systematic Review[J].JNeurosci Rural Pract,2018,9(4):478-486.
18 de Vries EE,van den Munckhof B,Braun KP,et al.Inflammatory mediators in human epilepsy:A systematic review and meta-analysis[J].Neurosci Biobehav Rev,2016,63:177-190.
19 Kovac S,Walker MC.Neuropeptides in epilepsy[J].Neuropeptides,2013,47(6):467-475.
2 No YJ,Zavanone C,Bielle F,et al.Medial temporal lobe epilepsy associated with hippocampal sclerosis is a distinctive syndrome[J].J Neurol,2017,264(5):875-881.
3 Gales JM,Jehi L,Nowacki A,et al.The role of histopathologic subtype in the setting of hippocampal sclerosis-associated mesial temporal lobe epilepsy[J].Hum Pathol,2017,63:79-88.
4 Miller-Delaney SF,Bryan K,Das S,et al.Differential DNAmethylation profiles of coding and non-coding genes define hippocampal sclerosis in human temporal lobe epilepsy[J].Brain,2015,138(Pt 3):616-631.
5韩春雷,葛燕,孟凡刚,等.海人酸致颞叶癫痫大鼠基因差异表达的研究[J].中华实验外科杂志,2014,31(3):567-569.
6 Griffin NG,Wang Y,Hulette CM,et al.Differential gene expression in dentate granule cells in mesial temporal lobe epilepsy with and without hippocampal sclerosis[J].Epilepsia,2016,57(3):376-385.
7 Bungenberg J,Surano N,Grote A,et al.Gene expression variance in hippocampal tissue of temporal lobe epilepsy patients corresponds to differential memory performance[J].Neurobiol Dis,2016,86:121-130.
8 Zubareva OE,Kovalenko AA,Kalemenev SV,et al.Alterations in mRNA expression of glutamate receptor subunits and excitatory amino acid transporters following pilocarpine-induced seizures in rats[J].Neurosci Lett,2018,686:94-100.
9 Mohrmann R,Dhara M,Bruns D.Complexins:small but capable[J].Cell Mol Life Sci,2015,72(22):4221-4235.
10 Chang S,Reim K,Pedersen M,et al.Complexin stabilizes newly primed synaptic vesicles and prevents their premature fusion at the mouse calyx of held synapse[J].J Neurosci,2015,35(21):8272-8290.
11 Lin MY,Rohan JG,Cai H,et al.Complexin facilitates exocytosis and synchronizes vesicle release in two secretory model systems[J].J Physiol,2013,591(10):2463-2473.
12 Landgraf I,Mühlhans J,Dedek K,et al.The absence of Complexin 3 and Complexin 4 differentially impacts the ON and OFF pathways in mouse retina[J].Eur J Neurosci,2012,36(4):2470-2481.
13 Mortensen LS,Park SJH,Ke JB,et al.Complexin 3 Increases the Fidelity of Signaling in a Retinal Circuit by Regulating Exocytosis at Ribbon Synapses[J].Cell Rep,2016,15(10):2239-2250.
14 Kurokawa A,Narukawa M,Ohmoto M,et al.Expression of the synaptic exocytosis-regulating molecule complexin 2 in taste buds and its participation in peripheral taste transduction[J].J Neurochem,2015,133(6):806-814.
15 Babai N,Sendelbeck A,Regus-Leidig H,et al.Functional Roles of Complexin 3 and Complexin 4 at Mouse Photoreceptor Ribbon Synapses[J].J Neurosci,2016,36(25):6651-6667.
16 Xue M,Stradomska A,Chen H,et al.Complexins facilitate neurotransmitter release at excitatory and inhibitory synapses in mammalian central nervous system[J].Proc Natl Acad Sci USA,2008,105(22):7875-7880.
17 Quintana-Pájaro LJ,Ramos-Villegas Y,Cortecero-Sabalza E,et al.The Effect of Statins in Epilepsy:A Systematic Review[J].JNeurosci Rural Pract,2018,9(4):478-486.
18 de Vries EE,van den Munckhof B,Braun KP,et al.Inflammatory mediators in human epilepsy:A systematic review and meta-analysis[J].Neurosci Biobehav Rev,2016,63:177-190.
19 Kovac S,Walker MC.Neuropeptides in epilepsy[J].Neuropeptides,2013,47(6):467-475.