【作者】 李刚；

【导师】 邓学军；

【作者基本信息】 华中科技大学 ， 神经病学， 2010， 硕士

【摘要】 目的:探讨匹罗卡品致癫痫状态下大鼠海马内肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)及星形胶质细胞的表达、增生规律,并探讨JAK/STAT信号转导通路在癫痫发作后对星形胶质细胞活化的影响。方法:应用腹腔注射氯化锂-匹罗卡品(PILO)的方法建立颞叶癫痫模型,应用JAK/STAT信号转导通路特异性抑制剂AG490进行腹腔注射建立干预模型组,应用生理盐水代替匹罗卡品进行腹腔注射建立对照模型组;用Western blot方法测定大鼠海马匀浆后TNF-α表达的变化,免疫荧光化学方法对照观察应用AG-490阻滞JAK/STAT通路后对大鼠海马TNF-α与星形胶质细胞活化的影响。结果: 1.大鼠行为学观察:生理盐水对照组雄性大鼠没有任何痫性发作。PILO致癫痫模型组有80.00%(32/40)的动物达Racine分级Ⅳ级以上发作;10.00%(4/40)的雄性大鼠死亡;10.00%(4/40)的雄性大鼠未达Racine分级Ⅳ级发作,模型成功率为90.00%;30天组中仍然有自发发作的有2只及25.00%(2/8)。AG490干预组雄性大鼠Ⅳ级以上发作的发生率为75.00%(30/40);死亡率为7.50%(3/40);17.50%(7/40)的雄性大鼠未达Ⅳ级发作,模型成功率为82.50%;30天时有Ⅳ级以上发作仅7只。达到V级发作占23只占76.67%(23/30)30天组中仍然有自发发作的有1只及12.50%(1/8),Ⅳ级及以下发作同PILO致癫痫模型组比较P<0.05有统计学意义;其他P>0.05,没有统计学意义。2.Western blotting检测发现PILO模型组中,TNF-α在癫痫发作3小时时即较生理盐水对照组开始增多,癫痫发作第3天时达到最高峰,之后逐渐下降,至癫痫第发作30天蛋白表达水平仍比正常略高,30天时有III级以上发作的比没有发作的要高;AG490干预组各时间点TNF-α表达均较PILO组明显减少。3h组、12h组分别和24h、3d组相比有显著性差异p<0.01,30d癫痫仍然有发作组和30d不发作组相比无显著差异p>0.013.双重免疫荧光结果:有黄绿色颗粒者为阳性染色,且可以看见阳性颗粒在星形胶质细胞内的空间分布,AG490腹腔注射建立干预后,阳性颗粒的星形胶质细胞明显减少,P<0.05,提示JAK/STAT信号转导通路可以促进星形胶质细胞活化。结论:应用氯化锂-匹罗卡品癫痫模型可以复制出与人类颞叶癫痫相似的癫痫病理学变化和行为学改变。在癫痫发作早期,海马区增生的星形胶质细胞活化可能是痫性发作导致神经元损伤的一种适应性反应,但是到了晚期,则有可能促进癫痫的进一步发作。AG490可阻断JAK/STAT信号转导通路进而抑制星形胶质细胞活化,同时也可以影响癫痫大鼠的行为学变化,提示AG490对癫痫的发作可能有一定的抑制作用,其机制可能与其抑制星形胶质细胞增生有关。更多还原

【Abstract】 Objective: To investigate the spatiotemporal distribution pattern of tumor necrosis factor (TNF-α) and reactive astrocytes in rats hippocampus following p Lithium-pilocarpine-induced seizures,and to infer the role of JAK/STAT signaling pathway in gliosis of rats with epilepsy.Methods: Temporal lobe epilepsy models were established on rats by intraperitoneal injection of Lithium-pilocarpine (PILO) , and specific JAK/STAT inhibitor-AG490 was used to set up pretreated models,and saline was injected in the normal control group. The expression pattern of TNF-αprotein and astrocytes was were measured and compared in each group with western blotting and double immunofluorescence assay before and after blockage of the JAK/STAT pathway in rat presented seizures hippocampus. Results:1. Behavior in rats: No seizures onset were observed in the Saline control group .Among the Lithium-pilocarpine -treated rats, 80.00%(34/40) of animals up to gradeⅣand above attack Racing , 10.10%(4/40) of male rats died, and 10.10%(4/40) of male rats below the gradeⅣclass attack Racing were expelled,Model of success rate was 90.00%, 30 days, the group still has two spontaneous seizures, and 25.00% (2 / 8).AG490 intervention groups of male rats,Ⅳlevel and above attack rate was 75.00%(30/40),mortality rate was 17.50%(7/40) and 2.50% were expelled, Accounted for 23 to V-class attack only 76.67% (23/30) 30-day group, there is still a spontaneous attack, and 12.50% (1 / 8),Ⅳlevel and below onset epilepsy model induced with PILO group P < 0.05; other P> 0.05.2. western blotting: PILO model group, tumor necrosis facto(rTNF-α)-alpha in seizures 3 hours after compared with saline control group,which began to increase, seizures reached a peak on day 3,then decreased gradually,The first 30 days of oneset of seizures to the protein level was still slightly higher than normal.AG490 intervention groups at each time point the expression of TNF-αsignificantly reduced compared with PILO group. 3h group, 12h respectively, and 24h, 3d group significantly different compared to p <0.01,30 d epilepsy still have seizures group and 30d do not attack groups was not significant( P> 0.05)3. Double Immunoflourescence Assay: A yellow-green particles were positive staining, positive cell can be seen in the astrocytes of the spatial distribution, AG490 intervention groups,positive granules of astrocytes were significantly reduced,(P<0.05). JAK/STAT pathway can promote the proliferation of astrocytes.Conclusion: limhium-pilocarpine epilepsy model on rats can reproduce human temporal lobe epilepsy similarly, whatever of behavior changes or pathological changes.In the early seizures,Proliferation of astrocytes in hippocampus cells can be regarded that seizures lead to neuronal injury in an adaptive response,but go there late ,may promote further seizures of epilepsy, AG490 can block the JAK/STAT signaling pathway and inhibit proliferation of astrocytes,And also can change the behavior and the reactive gliosis, AG490 reduce the oneset of epilepsy with the inhibition effects on the Proliferation of astrocytes.更多还原