【作者】 关立峰；

【导师】 王学峰；

【作者基本信息】 重庆医科大学 ， 神经病学， 2009， 博士

【摘要】 目的:观察ULBP2 (UL16结合蛋白2)和隆文菌素A (STRN)在耐药性颞叶癫痫患者脑组织中的表达,探讨其在耐药性颞叶癫痫发病中的意义。资料和方法:试验组42例均为临床确诊的耐药性癫痫患者,发作类型符合1981年国际抗癫痫联盟(ILAE)有关癫痫发作分类的标准。其中男26例,女16例,年龄15～56岁,平均26.18±9.60(x±s)岁;病程3～30年,平均为12.88±6.49( x±s)年;发作类型中部分继发全身强直、阵挛或强直-阵挛性发作23例,复杂部分性发作6例,有多种发作类型(单纯部分性、强直性或强直-阵挛性发作)13例。术后获颞叶标本34例,海马标本8例。对照组共有20例,全部为在我院或第三军医大学新桥医院神经外科行手术减压或清创患者的颞叶和海马脑组织,12例颞叶,8例海马。其中男7例,女5例,年龄16-44岁,平均23.05±9.17岁(X+SD)。结果:1.基因芯片结果耐药性癫痫组脑组织ULBP2蛋白的基因表达上调,与正常对照组间比较有明显差异(Ratio=4.180)。耐药性癫痫组脑组织STRN蛋白的基因表达下调,与正常对照组间比较有明显差异(Ratio=0.384)。2.免疫组化ULBP2主要在神经元和神经胶质细胞的胞膜表达,阴性对照表达极少或无表达。耐药性癫痫颞叶脑组织的A值(AOD值)值为0.0518±0.0232 (x±s),对照组为0.0206±0.0011(x±s),两组方差不起,校正后t’检验结果显示两组间差异有统计学意义(t’=4.969,p<0.05)。耐药性癫痫组海马的AOD值为0.0509±0.0048(x±s),对照组为0.0198±0.0114( x±s),两组间有显著差异(t=2.528, p<0.05)。STRN正常主要在神经元和神经胶质细胞的胞浆表达,实验组表达极少或无表达。耐药性癫痫颞叶脑组织的A值(AOD值)值为0.3108±0.1087 (x±s),对照组为0.4210±0.1238(x±s),两组方差不起,校正后t’检验结果显示两组间差异有统计学意义(t’=4.858,p<0.05)。耐药性癫痫组海马的AOD值为0.2706±0.0038(x±s),对照组为0.3952±0.0107( x±s),两组间有显著差异(t=2.416, p<0.05)。3.免疫荧光在对照组ULBP2蛋白表达极少,而在实验组中ULBP2基因的蛋白表达产物比对照组相同部位的脑组织中表达明显增加。在对照组STRN蛋白表达明显,而在实验组中STRN蛋白表达产物比对照组相同部位的脑组织中表达明显减少。4.免疫印记法ULBP2蛋白条带在对照组明显减弱,反映出实验组中ULBP2基因的蛋白表达产物比对照组相同部位的脑组织中表达明显增加。耐药性癫痫颞叶脑组织灰度值0.9804±0.1401,对照组灰度值0.5914±0.2197,两组间差异有统计学意义(t=2.7633,p<0.05)。耐药性癫痫海马脑组织灰度值0.9832±0.1508,对照组灰度值0.5866±0.1974,两组间差异有统计学意义(t=2.7838,p<0.05)。STRN蛋白条带在实验组明显减少,反映出实验组中STRN基因的蛋白表达产物比对照组相同部位的脑组织中表达明显减少。耐药性癫痫颞叶脑组织灰度值0.3318±0.1101,对照组灰度值0.5846±0.1024,两组间差异有统计学意义(t=2.6613,p<0.05)。耐药性癫痫海马脑组织灰度值0.2786±0.0308,对照组灰度值0.5686±0.1214,两组间差异有统计学意义(t=2.6808,p<0.05)。结论:耐药性颞叶癫痫患者颞叶脑组织中ULBP2基因及蛋白产物表达增高可能与耐药性颞叶癫痫发病中的免疫异常机制有重要关联,甚至可能是关键因素。高表达的ULBP2可通过与NK细胞、T细胞、活性巨噬细胞、CD8细胞表面的NKG.2D受体特异性结合,引起复杂的系列免疫反应,并导致许多自身免疫抗体的大量产生和灭活减少,从而诱发癫痫或是使癫痫发作加重难以控制。它很可能为耐药性颞叶癫痫的免疫治疗提供新的靶点。耐药性颞叶癫痫患者颞叶脑组织中STRN表达明显降低及其引发的雌激素信号转导和内源性一氧化氮合成减弱在耐药性颞叶癫痫发病中可能有重要作用。更多还原

【Abstract】 Objective: To study the expression of genes and protein of UL16 binding protein 2 and STRN in the brain tissue of patients with drug-refractory epilepsy , and to explore the possible role in drug-refractory epilepsy and the clinical value.Materials and Methods: The 42 cases with refractory epilepsy included in this study were obtained from the files of the Departments of Neurosurgery of the following hospitals: the First Affiliated Hospital of Chongqing Univerisyt of Medical Sciences, Beijing Tiantan Hospital, Xuanwu Hospital of the Capital University of Medical Sciences, and Xinqiao Hospital of the Third Military Medical Univeristy. All the patients involved in our study group underwent the surgical resection of temporal lobe tissue. Before surgery, informed consents were obtained for the use of human brain tissues for research, and the study was approved by the local Ethic Committee. Two neuropathologists reviewed all the cases independently. The diagnosis of seizure type was confirmed according to the 1981 International Classification of Epileptic Seizures of the International League Against Epilepsy.The 20 controls were obtained from the files of the neurosurgery department of the first affiliated hospital of Chongqing University of Medical Sciences. These samples were comprised of temporal neocortical tissues adjacent of the lesion. All patients were diagnosed by pathological tests to have been suffered brain trauma. the two neurophthologists both reviewed these cases to confirm that they had no history of seizures or other neurological disorders as controls.Gene-chip, immunohistochemistry, immunofluorescence and Western blotting were used to test expression of ULBP2 protein and STRN protein in the surgically removed brain tissue of patients with drug-refractory epilepsy from the brain bank of our department(n=42), and the results were compared with that of normal controls (n=20)。The Computer Graphic Analysis System is used to test the average optical density (AOD) which is expressed as mean±SD( x±s)and tested via t-test. P value less than 0.05 as the level of significance.Results1.Gene chip: There was significant difference between the control group and the pharmacoresistance group (Ratio=4.180). The expression of SCN8A gene was shown to be increased in the brain tissues of the pharmacoresistance epilepsy group. There was significant difference between the control group and the pharmacoresistance group (Ratio=0.384). The expression of STRN gene was shown to be decreased in the brain tissues of the pharmacoresistance epilepsy group. 2. Immunohistochemistry : In human temporal cortex of patients with epilepsy, ULBP2 immunoreactivity was consistently observed in all cases and was confined mainly to neurons and glial cells, whereas faint immunoreactivity was apparent in the control brain tissues. Four visual field images were obtained randomly in every section by TC-FY-2050 pathology system acquisi-tioned image and were automatically analyzed by Motic Med 6.0 CMIAS pathology image analysis system. AOD value in epileptic temporal cortex tissue tissues was determined 0.0518±0.0232 ( x±s), and 0.0206±0.0011 ( x±s) in the control. AOD value in hippocampus of pharmacoresistance epilepsy group was determined 0.0509±0.0048( x±s), and 0.0198±0.0114 ( x±s) in the control. ANOVA analysis shows significantly up-regulated ULBP2 in epileptic tissue than the control (P<0.05).In control brain tissues, STRN immunoreactivity was consistently observed in all cases and was confined mainly to neurons and glial cells, whereas faint immunoreactivity was apparent in control brain tissues. Four visual field images were obtained randomly in every section by TC-FY-2050 pathology system acquisi-tioned image and were automatically analyzed by Motic Med 6.0 CMIAS pathology image analysis system. AOD value in epileptic temporal cortex tissue tissues was determined 0.3108±0.1087 ( x±s), and 0.4210±0.1238 ( x±s) in the control. AOD value in hippocampus of pharmacoresistance epilepsy group was determined 0.2706±0.0038 ( x±s), and 0.3952±0.0107 ( x±s) in the control. ANOVA analysis shows significantly down-regulated STRN in epileptic tissue than the control (P<0.05).3. Immunofluorescence analysis: In the temporal lobe cortex and hippocampus, strong immunoreactivity for the staining of ULBP2 protein was observed. There were more obvious in the pharmacoresistance epilepsy group than in control group.In the temporal lobe cortex and hippocampus, faint immunoreactivity for the staining of STRN protein was observed. There were less obvious in the pharmacoresistance epilepsy group than in control group.4. Western blot: Immunoblot was performed with ULBP2 antibody, the protein was detected by immunoblot as a band at 29kDa. Strongly stained bands were present in all samples of epileptic tissue whereas relative faint expression of the ULBP2 was observed in the control group. Another band ofβ-actin as positive control at 42 kDa also was observed correspondently in every channel. The ratio of optical density of ULBP2 andβ-actin was taken as variance calculated between two groups. Optical density value in Epileptic temporal cortex tissue was 0.9804±0.1401 and 0.5914±0.2197 ( x±s) in the control tissue. Optical density value in Epileptic hippocampus tissue was 0.9832±0.1508 and ( x±s)0.5866±0.1974 in the control tissue.The result shows a significant difference between epileptic tissue and the control group (P<0.05) .Immunoblot was performed with STRN antibody, the protein was detected by immunoblot as a band at 110kDa. faint stained bands were present in all samples of epileptic tissue whereas relative strongly expression of the STRN was observed in the control group. Another band ofβ-actin as positive control at 42 kDa also was observed correspondently in every channel. The ratio of optical density of STRN andβ-actin was taken as variance calculated between two groups. Optical density value in Epileptic temporal cortex tissue was 0.3318±0.1101 and 0.5846±0.1024 ( x±s) in the control tissue. Optical density value in Epileptic hippocampus tissue was 0.2786±0.0308 ( x±s)and 0.5686±0.1214 in the control tissue.The result shows a significant difference between epileptic tissue and the control group (P<0.05) .Conclusions:Our work showed that a increase in ULBP2 cDNA and protein level may be involved in the pathophysiology of refractory epilepsy which suggested that the ULBP2 may play a key role in the epilepsy immune disorder,so bring some promising immune targets toward the therapy of refractory epilepsy. Additional studies will be required to elucidate the mechanism by which ULBP2 affects brain function in refractory epilepsy.Our work also showed that a decrease in STRN cDNA and protein level may be involved in the pathophysiology of refractory epilepsy which suggested that the STRN may be associated with impairment of the brain protection cased by endogenous NO in refractory epilepsy,so bring some promising targets toward the therapy of refractory epilepsy. Additional studies will be required to elucidate the mechanism by which STRN affects brain function in refractory epilepsy.更多还原