【作者】 肖飞；

【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】 第一部分:耐药性癫痫患者脑组织中N-WASP及上、下游基因和蛋白表达的研究目的:研究神经元威斯科特-奥尔德里奇综合征蛋白(neuronal Wiskott-Aldrich syndrome protein,N-WASP)及上游分子细胞分裂周期42 GTP结合蛋白(cell division cycle 42 GTP-binding protein, Cdc42),下游分子肌动蛋白相关蛋白2/3 (actin-related protein 2/3, Arp2/3)在耐药性癫痫患者脑组织中的表达,探讨N-WASP在耐药性癫痫发病机制中的作用。方法:基因芯片扫描耐药性癫痫患者脑组织发现N-WASP显著升高后,分别用蛋白印迹、免疫组化及免疫荧光研究40例耐药性癫痫患者脑组织中N-WASP的表达,并分别用蛋白印迹、免疫组化及荧光研究其上、下游分子Cdc42、Arp2/3的表达,并与对照组进行比较。结果:基因芯片扫描显示N-WASP在耐药性癫痫组中明显增高(Cy5/Cy3=3.021),蛋白印迹示耐药性癫痫组脑组织中N-WASP相对灰度值0.9030±0.0492 (x±s),对照组脑组织为0.4320±0.0727 (x±s) (P<0.05)。免疫组化示N-WASP在耐药性癫痫患者组的OD值为0.384±0.072,比对照组0.143±0.046明显升高(P<0.05),免疫荧光与免疫组化结果一致。Cdc42在耐药性癫痫和对照组中的OD值分别为0.032±0.016,0.020±0.005 (P<0.05)。Arp2/3免疫印迹检测其在耐药性癫痫组和对照组中的灰度值分别为0.7765±0.0230,0.4986±0.0823,癫痫组明显增高(P<0.05)。以上结果显示N-WASP、Cdc42和Arp2/3在耐药性癫痫患者脑组织中表达显著上调。结论:N-WASP及其上游Cdc42,下游Arp2/3在耐药性颞叶癫痫患者脑组织中表达增高,提示该通路在耐药性颞叶癫痫的发生机制中可能有重要作用。第二部分耐药性癫痫患者脑脊液中生物标记物的筛选目的:研究耐药性颞叶癫痫患者脑脊液中差异蛋白的表达,通过与对照组比较,寻找耐药性癫痫患者脑脊液中的癫痫生物标记物。方法:运用双向凝胶电泳对两组脑脊液进行分离,图像分析后对差异点行液相色谱电喷雾串联质谱鉴定并进行数据库查询。对鉴定出来的蛋白质通过免疫印迹进行验证。结果:两组脑脊液中8个蛋白质点有显著差异表达(p<0.05) ,其中维生素D结合蛋白(vitamin D-binding protein, DBP)在耐药性癫痫患者中增高,而色素上皮衍生因子(pigment epithelium-derived factor, PEDF),组织蛋白酶D (cathepsin D, CTSD),gelsolin,载脂蛋白J (apolipoprotein J,apoJ ), Ras蛋白特异性鸟嘌呤核苷酸释放因子1(Ras protein-specific guanine nucleotide-releasing factor 1, RASGRF1 ), Fam3c和超氧化物歧化酶1 (superoxide dismutase 1,SOD1)在耐药性患者脑脊液中减少。另外6个蛋白点只在癫痫患者脑脊液胶图上出现,分别是四连结素(tetranectin,TN), talin-2,载脂蛋白E (apolipoprotein E, apoE),免疫球蛋白轻链lambda (immunoglobulin lambda light chain, IGL @ ),免疫球蛋白轻链kappa可变区1-5(immunoglobulin kappa variable light chain 1-5, IGKV1– 5)和胶原C肽链内切酶增强子1 (procollagen C-endopeptidase enhancer 1, PCOLCE)。免疫印迹分析DBP、SOD1和talin-2的表达证实了质谱鉴定的结果。结论:我们的研究结果提示耐药性癫痫患者脑脊液中存在着异常表达的癫痫生物标记物,该结果可能帮助我们更好的了解耐药性癫痫患者发病的病理生理机制。更多还原

【Abstract】 PART ONE: EXPRESSION OF N-WASP AND ITS UPSTREAM AND DOWNSTREAM GENE AND PROTEIN EXPRESSION IN BRAIN TISSUE OF PATIENTS WITH DRUG-RESISTANT EPILEPSYObjective To investigate the expression of neuronal Wiskott-Aldrich syndrome protein (N-WASP), and its upstream elements cell division cycle 42 GTP-binding protein (Cdc42), downstream molecules actin-related protein 2/3 (Arp2 / 3) in the brain tissues obtained from patients with drug-resistant epilepsy and discuss their significances in epileptogenesis.Methods cDNA microarray scanning has shown that N-WASP significantly increased in brain tissue of patients with drug-resistant epilepsy. Western blot, immunohistochemistry and immunofluorescence were used to evaluate the expression of N-WASP, immunohistochemistry and immunofluorescence were used to evaluate the expression of its upstream regulator Cdc42. Western blot was used to detect its downstream effector Arp2/3 expression in 40 cases of drug-resistant epilepsy, respectively. Comparison was made with the control group.Results cDNA microarray result showed the Cy5/Cy3 ratio of N-WASP was 3.021. Western blot gray value was 0.9030±0.0492 ( x±s) in drug-resistant epilepsy group and 0.4320±0.0727 ( x±s) in control group (P<0.05). The immunohistochemistry OD value of epilepsy group was 0.384±0.072 ( x±s), and 0.143±0.046 ( x±s) for control group (P <0.05). The data of immunofluorescence analysis complied with the outcome of immunohistochemistry. OD values of Cdc42 in drug-resistant epilepsy group and control group were 0.032±0.016 ( x±s), 0.020±0.005 ( x±s), respectively (P <0.05). Gray value of Arp2 / 3 was 0.7765±0.0230 ( x±s) in case group, compared to 0.4986±0.0823 ( x±s) in control group (P <0.05). These results indicated that N-WASP, Cdc42 and Arp2 / 3 were upregulated in brain tissue of patient with drug-resistant epilepsy.Conclusions Overexpression of N-WASP and its upstream Cdc42, downstream Arp2 / 3 in patients with drug-resistant temporal lobe epilepsy, suggesting that this pathway may play an important role in the mechanisms of drug-resistant epilepsy. PART TWO: BIOMARKER SCREENING OF CEREBROSPINAL FLUID IN PATIENTS WITH DRUG-RESISTANT EPILEPSYObjective To investigate the proteomic changes of cerebrospinal fluid (CSF) in patient with drug-resistant epilepsy in order to discover cerebrospinal fluid biomarkers of epilepsy.Methods Two-dimensional gel electrophoresis followed by liquid chromatography electrospray ionization tandem mass spectrometry were used, these proteins were identified by database searching. Expression of some proteins was validated by western blot.Results Eight protein spots showed significant differential expression (p<0.05): vitamin D-binding protein (DBP) was elevated in the CSF of TLE patients whereas pigment epithelium-derived factor (PEDF), cathepsin D (CTSD), gelsolin, apolipoprotein J (apoJ), Ras protein-specific guanine nucleotide-releasing factor 1 (RASGRF1), Fam3c, and superoxide dismutase 1 (SOD1) were decreased in the CSF of TLE patients. Additional six protein spots presented only in the CSF of epilepsy patients were identified as tetranectin (TN), talin-2, apolipoprotein E (apoE), immunoglobulin lambda light chain (IGL@), immunoglobulin kappa variable light chain 1-5 (IGKV1-5), and procollagen C-endopeptidase enhancer 1 (PCOLCE). Expression of DBP、SOD1and talin-2 was validated by western blot.Conclusions Our results suggest that there exsist disease specific biomarkers of cerebrospinal fluid in patients with intractable epilepsy, which may provide better understanding of the pathophysiologic mechanisms underlying epileptogenesis.更多还原