颞叶癫痫相关突触后致密物蛋白质的蛋白质组学筛选研究

目的 筛选颞叶癫痫(TLE)相关突触后致密物(PSD)蛋白分子.方法 建立氯化锂-匹罗卡品颞叶癫痫大鼠模型;收集颞叶癫痫组(TLE组)、非颞叶癫痫组(非TLE组)以及正常对照组(Norm)3组的新鲜全脑组织,联合应用蔗糖密度梯度离心和膜顺序提取技术提取PSD;进行双向凝胶电泳,并通过PDQuest图像分析软件比较分析,筛选TLE组特异性和差异性表达的蛋白质点;选取部分差异蛋白质点进行取MALDI-TOF-MS质谱鉴定;应用Western验证部分所鉴定蛋白质在PSD中的表达状况.结果 与Norm和非TLE组比较,TLE组中存在40个差异表达蛋白质点,表达上调的主要有热休克蛋白27(HSP-27)、果糖二磷酸醛缩酶(FBA)、肌酸激酶(CK)、甲状腺受体相互作用蛋白6(TRIP6)、髓鞘碱性蛋白S(MBP)、LIM结构域(LIM);表达下调的主要有细胞骨架蛋白、肽酰-脯氨酰-顺反式异构酶(PPIase)、分选连接蛋白3(SNX3)、乌头酸水合酶(ACO)、甘油醛三磷酸脱氢酶(GAPDH)、琥珀酸辅酶A连接酶(SCOAL).HSP27和微管蛋白α、SNX3在TLE、非TLE和Norm 3组PSD中均免疫染色阳性.结论 TLE组PSD蛋白质差异性表达可能是脑损伤后可塑性反应的结果,但其表达量改变的程度可能与颞叶癫痫形成有关,可视为促/抗TLE形成的重要候选分子.

Proteomic screening of postsynaptic density proteins related with temporal lobe epilepsy

Objective To explore the postsynaptic density (PSD) proteins related with the development of temporal lobe epilepsy (TLE). Methods Five SD rats were injected intra-peritoneally with lithium chloride and then pilocarpine twice to establish epilepsy models. At last 24 rats developed TLE,and 12 developed non-TLE. Then rats underwent intraperitoneal injection of normal saline (Norm group). Thirty days after the appearance of epilepticism the rats were decapitated with their brains taken out. The PSD proteins were extracted and purified by using sucrose gradient centrifugation and membrane sequence extraction,isolated by using two-dimensional gel electrophoresis. PDQuest software was used to screen the specifically and differentially expressed protein spots. Partial differentially expressed PSD protein spots were selected and identified by MALDI-TOF-MS. Several identified proteins were detected in the PSD fraction by Western blotting. Results Compared with the non-TLE and Norm groups,there were 40 ifferential protein spots in the TLE group. The expression levels of heat shock protein-27 (HSP-27),fructose-bisphosphate aldolase A (FBA),creatine kinase (CK),thyroid receptor-interacting protein 6 (TRIP6),myelin basic protein S (MBP) ,and LIM domain were up-regulated,but the expression levels of tubulin,actin,interoexin-alpha,peptidyl-prolyl cis-trans isomerase (PPIase) ,sorting nexin 3 (SNX3) ,aconitate hydratase (ACO) ,glyceradehydea-3-phosphate ehydrogenase (GADPH),and succinate-coenzyme A ligase (SCOAL) were down-regulated in the TLE group. The HSP27,tubulin-alpha,and SNX3 were in the PSD gels were immunostaining positive in the 3 groups. Conclusion The differential expression of PSD proteins in TLE may be due to injury induced neural plasticity. But the degree thereof may contribute to the development of TLE. These identified proteins can be regarded as important candidates for or against the development of TLE.