非病灶性难治性颞叶癫痫的相关基因研究
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摘要
本课题的目的在于从非病灶性颞叶癲痫入手，筛选颞叶癫痫相关基因表达谱，并通过对表达谱筛选所发现的差异目标基因进行验证，从分子水平探究非病灶性颞叶癲痫可能的发病机制，以期为难治性颞叶癲痫的诊断与治疗开辟新途径。第一部分应用新兴的分子生物学技术——cDNA微列阵技术，利用含有18000个cDNA克隆的微列阵对难治性非病灶性颞叶癲痫患者手术切除颞叶组织标本与对照间行基因表达谱分析研究；结果显示：两份颞叶癲痫样品中表达趋势一致的己知基因主要包括：免疫相关因子、离子通道相关因子、信号转导相关基因、细胞增殖周期因子、细胞凋亡相关基因等，在颞叶癫痫发生发展各个阶段均有基因的参与，是一个复杂的调控过程。第二部分应用半定量RT-PCR方法，根据前期实验所获得的结论，检测12例非病灶性难治性颞叶癫痫患者和10例对照颞叶组织中编码内向整流钾离子通道kir2.3亚家族的KCNJ4 mRNA表达水平的差异；结果发现：与对照组相比，颞叶癫痫组KCNJ4 mRNA表达水平明显降低，差异具有显著统计学意义(P＜0.05)。第三部分应用免疫组化以及Western blot方法，分析了KCNJ4基因所编码的内向整流钾通道kir2.3蛋白在非病灶性难治性颞叶癫痫患者与对照脑组织中的表达差异，结果显示：kir2.3通道蛋白在颞叶癫痫患者颞叶组织中的表达水平明显低于对照组，差异有显著统计学意义(P＜0.05)。综合上述结果，我们推测，KCNJ4基因表达水平的异常可能使其编码的内向整流钾离子通道kir2.3亚家族的蛋白表达水平下调，从而干扰了神经细胞膜的稳定性、同时使得胶质细胞对过度钾离子负荷的缓冲能力下降；内向整流钾通道表达水平的变化，可能影响了其维持细胞兴奋性平衡作用，最终导致痫样放电的发生。由此，我们进一步推测，kir2．3通道亚家族可能是一个抗癫痫药物的新的理想作用位点。
The aim of this study is to survey gene expression profiles in nonlesional refractory temporal lobe epi lepsy (TLE) , to further verify the difference of gene expression, then to evaluate the possible molecular pathogenesis of this kind of epilepsy and can help to supply a new way for diagnosis and therapy. In the first part, the difference of gene expression was studied in 2 cases of TLE samples and 1 mixed sample from normal control tissue by methods of cDNA microarray consisting of 18000 genes. The results showed that, those different expressed genes in two TLE samples altered with the same tendency including: immune correlative genes, ionic channel transportation genes, signal conduction genes, mitochondral function genes and so on. Gene took part in all the phases during occurrence and progress of TLE, it was quite a complex procedure. In the second part, on the basis of prophase results , KCNJ4 mRNA level was tried to be examined in the temporal lobe tissue in 12 TLE patients and 10 trauma controls by
RT-PCR. Compared with trauma controls, KCNJ4 mRNA level of TLE patients decreased statistically ( P<0.05 ). In the third parts, immunohistochemistry and western blotting were used to detect the changes of kir2.3 channel protein in TLE patients and controls. The results showed that, kir2.3 protein level of TLE patients also decreased statistically (P<0.05), compared with controls. From the researches of all above parts, we concluded that the change of KCNJ4 mRNA level and the decreased level of kir2. 3 channel protein in TLE patients could result in disturbing the stability of neuronal cell membrane as well as reducing the amortization ability to super-potassium loading of glial cell, affecting the maintenance of cell excitability equilibrium and finally cause epileptiform discharging. Furthermore, we evaluate that potassium channel kir2.3 subunit could be appropriate role site for those antiepileptic drugs.
The aim of this study is to survey gene expression profiles in nonlesional refractory temporal lobe epi lepsy (TLE) , to further verify the difference of gene expression, then to evaluate the possible molecular pathogenesis of this kind of epilepsy and can help to supply a new way for diagnosis and therapy. In the first part, the difference of gene expression was studied in 2 cases of TLE samples and 1 mixed sample from normal control tissue by methods of cDNA microarray consisting of 18000 genes. The results showed that, those different expressed genes in two TLE samples altered with the same tendency including: immune correlative genes, ionic channel transportation genes, signal conduction genes, mitochondral function genes and so on. Gene took part in all the phases during occurrence and progress of TLE, it was quite a complex procedure. In the second part, on the basis of prophase results , KCNJ4 mRNA level was tried to be examined in the temporal lobe tissue in 12 TLE patients and 10 trauma controls by
RT-PCR. Compared with trauma controls, KCNJ4 mRNA level of TLE patients decreased statistically ( P<0.05 ). In the third parts, immunohistochemistry and western blotting were used to detect the changes of kir2.3 channel protein in TLE patients and controls. The results showed that, kir2.3 protein level of TLE patients also decreased statistically (P<0.05), compared with controls. From the researches of all above parts, we concluded that the change of KCNJ4 mRNA level and the decreased level of kir2. 3 channel protein in TLE patients could result in disturbing the stability of neuronal cell membrane as well as reducing the amortization ability to super-potassium loading of glial cell, affecting the maintenance of cell excitability equilibrium and finally cause epileptiform discharging. Furthermore, we evaluate that potassium channel kir2.3 subunit could be appropriate role site for those antiepileptic drugs.
引文
1. Regesta G, Tanganelli P. Temporal lobe epilepsy of adult age of possible idiopathic, nature. Seizure, 2002 Mar, 11(2) :131-135
2．吴洵昳，洪震．颞叶癫痫的基因研究进展．神经疾病与精神卫生，2002，2(4)：250-251
3．吴逊，主编。神经病学：癫痫和发作性疾病．北京：人民军医出版社，2001：1
4. Serratosa JM. Idiopathic epilepsies with a complex mode of inheritance. Epilepsia, 1999,40 Supple 3:12-16
5. Ben Ari Y. Cell death and synaplic reorganizations produced by seizures. Epilepsia, 2001,42 Supple 3:5-7
6．王雄清，吴三林．参与细胞生长和凋亡的MAPK级联．乐山师范学院学报，2000，3：69-73
7．胡智，曹亚．MAPK／JNK信号传导通路研究进展．国外医学分子生物学分册，2002，24(4)：222-225
8. Schauwecker PE. Seizure-induced neuronal death is associated with induction of c-jun N-terminal kinase and is dependent on genetic background. Brain Res, 2000 Nov 24:116-128
9. Perosa SR, Porcionatto MA, Cukiert i, et al. Glycosaminoglycan levels and proteoglycan expression are altered in the hippocampus of patients with mesial temporal lobe epilepsy. Brain Res Bull, 2002 Sep 15,58(5):509-516
10. Perosa SR, Porcionatto MA, Cukiert A, et al. Extracellular matrix components are altered in the hippocampus, cortex, and cerebrospinal fluid of patients with mesial temporal lobe epilepsy. Epilepsia, 2002, 43 Supple 5:159-161
11. Ozkara C, Altintas A, Yilmaz E, et al. An association between mesial temporal lobe epilepsy with hippocampal sclerosis and human leukocyte antigens. Epilepsia, 2002 Jul, 43(7):777
12. Roll P, Szepetowski P. Epilepsy and ionic channels. Epileptic Disorder, 2002,3:165-172
13．关文标，佴玉，罗祖明等．钾离子通道与癫痫．国外医学神经病学神经外科学分册，2002，29(5)：442-444
14．张乐之，开丽，谭娅．平滑肌内向整流钾通道．生命科学研究，2002，6(1)：70
15. Kaupmann K, Schuler V, Mosbacher, T, et al. Human gamma-aminobutyric acid type Breceptors are differentially expressed and regulate inwardly recitifying K+ channels. Proc Natl Acad Sci USA, 1998,95(25):14491
16．洪燕，程义勇．脑内金属硫蛋白表达及其功能的研究进展．国外医学医学地理学分册，2002，23(1)：1-3
2．吴洵昳，洪震．颞叶癫痫的基因研究进展．神经疾病与精神卫生，2002，2(4)：250-251
3．吴逊，主编。神经病学：癫痫和发作性疾病．北京：人民军医出版社，2001：1
4. Serratosa JM. Idiopathic epilepsies with a complex mode of inheritance. Epilepsia, 1999,40 Supple 3:12-16
5. Ben Ari Y. Cell death and synaplic reorganizations produced by seizures. Epilepsia, 2001,42 Supple 3:5-7
6．王雄清，吴三林．参与细胞生长和凋亡的MAPK级联．乐山师范学院学报，2000，3：69-73
7．胡智，曹亚．MAPK／JNK信号传导通路研究进展．国外医学分子生物学分册，2002，24(4)：222-225
8. Schauwecker PE. Seizure-induced neuronal death is associated with induction of c-jun N-terminal kinase and is dependent on genetic background. Brain Res, 2000 Nov 24:116-128
9. Perosa SR, Porcionatto MA, Cukiert i, et al. Glycosaminoglycan levels and proteoglycan expression are altered in the hippocampus of patients with mesial temporal lobe epilepsy. Brain Res Bull, 2002 Sep 15,58(5):509-516
10. Perosa SR, Porcionatto MA, Cukiert A, et al. Extracellular matrix components are altered in the hippocampus, cortex, and cerebrospinal fluid of patients with mesial temporal lobe epilepsy. Epilepsia, 2002, 43 Supple 5:159-161
11. Ozkara C, Altintas A, Yilmaz E, et al. An association between mesial temporal lobe epilepsy with hippocampal sclerosis and human leukocyte antigens. Epilepsia, 2002 Jul, 43(7):777
12. Roll P, Szepetowski P. Epilepsy and ionic channels. Epileptic Disorder, 2002,3:165-172
13．关文标，佴玉，罗祖明等．钾离子通道与癫痫．国外医学神经病学神经外科学分册，2002，29(5)：442-444
14．张乐之，开丽，谭娅．平滑肌内向整流钾通道．生命科学研究，2002，6(1)：70
15. Kaupmann K, Schuler V, Mosbacher, T, et al. Human gamma-aminobutyric acid type Breceptors are differentially expressed and regulate inwardly recitifying K+ channels. Proc Natl Acad Sci USA, 1998,95(25):14491
16．洪燕，程义勇．脑内金属硫蛋白表达及其功能的研究进展．国外医学医学地理学分册，2002，23(1)：1-3