【作者】 蒋国会；

【导师】 王学峰；

【作者基本信息】 重庆医科大学 ， 神经病学， 2013， 博士

【摘要】 第一部分IGF-1在颞叶癫痫患者及点燃大鼠脑组织中的表达目的：研究IGF-1在难治性颞叶癫痫（TLE）患者颞叶皮质中的表达，及IGF-1在化学点燃模型即匹罗卡品（PILO）诱导颞叶癫痫模型和戊四氮（PTZ）急性点燃模型海马脑组织中的动态表达规律。方法：人脑组织标本源自课题组的癫痫脑库，从中随机抽取24例难治性TLE患者颞皮质标本，12例对照组标本。酶联免疫吸附实验（enzyme-linked immunosorbent assay, ELISA）用于检测检测IGF-1蛋白表达，荧光定量PCR（quantitative real time PCR, qRT-PCR）技术检测IGF-1mRNA表达水平。用ELISA和qRT-PCR检测成年雄性SD大鼠IGF-1表达，动物随机分为正常对照组（n=5）和癫痫组（SE后6h组、24h组、7d组、21d组、60d组,各组n=5），经腹腔注射PILO诱导癫痫持续状态（status epilepticus, SE），构建SE后颞叶癫痫动物模型。用PTZ大鼠急性点燃模型验证急性期IGF-1的表达，分6h组（n=5）和24h组（n=5）。结果：IGF-1mRNA及蛋白在TLE患者颞叶皮质中的表达较非癫痫对照组颞叶皮质表达水平明显增高，IGF-1mRNA表达的相对量对照组为1.19±0.34，TLE组为2.46±1.48；IGF-1蛋白对照组为235.75±50.54pg/mg，TLE组为311.58±101.79pg/mg，（P＜0.05）。PILO模型鼠IGF-1mRNA相对水平在SE后24h、21d和60d三个时间点的表达显著增高，对照组均值为1.03±0.05，24h组为2.15±0.46，21d组为2.69±0.55，60d组为3.57±0.64，差异有统计学意义（P＜0.05）；ELISA结果显示IGF-1蛋白水平在PILO模型癫痫急性期（SE后6h和24h）和SRS后（SE后21d和60d）IGF-1蛋白水平表达均显著增高（P＜0.05）；对照组为9.16±0.83， SE后6h组16.04±2.24，24h组19.98±2.18，21d组13.41±2.28，60d组为13.26±1.9（ng/mg蛋白）；PTZ模型GTCS6h和24h后IGF-1蛋白表达水平也显著增高（P＜0.05），分别为14.05±2.03和13.96±3.58（ng/mg蛋白）。结论：在颞叶癫痫患者和动物模型脑组织中IGF-1表达均明显增加，SE后IGF-1基因和蛋白的高表达可能参与了癫痫的发生发展过程。第二部分癫痫研究新发现：IGF-1及其受体信号通路调控癫痫活动目的：通过大鼠活体侧脑室注射重组人胰岛素样生长因子（rhIGF-1），腹腔注射特异性IGF-1受体抑制剂（picropodophyllin,PPP），观察其对PILO和PTZ模型鼠癫痫活动的影响。大鼠活体给予IGF-1受体下游信号通路MAPK-ERK抑制剂U0126和PI3K-AKT抑制剂LY294002，分别观察其对PTZ模型鼠癫痫活动的影响。方法：所有动物麻醉后经立体定位侧脑室埋置微量注射导管，术后恢复一周，动物在清醒自由活动状态下药物干预，造模后观察大鼠行为学变化。分组：对照组（n=13），侧脑室注射等量生理盐水；IGF-1组（n=13），侧脑室注射rhIGF-110μg；PPP组（n=15），侧脑室注射（NS），腹腔注射PPP20mg/kg体重；干预后1h分别腹腔注射PILO诱导SE。PTZ模型采用同样的分组及干预方法（各组n=10）。PILO模型观察SE潜伏期，4级以上SE发作比率，最大发作评分（Racine score）和急性期死亡率。PTZ模型观察惊厥发作潜伏期和发作比率。两组均记录脑电活动（n=3）。IGF-1下游信号干预分为对照组，U0126组（5μΜ, i.c.v.），LY294002组（20μΜ, i.c.v.），U0126+LY294002组（U01265μM+LY29400220μM, i.c.v.）（各组n=10）。干预后1h分别腹腔注射PTZ造模，观察惊厥发作潜伏期和发作比率。结果：PILO模型对照组SE潜伏期均值为28.89±7.96(min)，IGF-1组为19.58±8.05(min)，PPP组为38.63±13.06(min)；用ANOVA多种样本均数间比较显示与对照组比较，IGF-1组潜伏期明显缩短，PPP组潜伏期明显延长（P＜0.05）。大鼠IV级以上发作比率显示：对照组为9/12，IGF-1组为12/13，PPP组为8/15，IGF-1组和PPP组比较有显著统计学差异。对照组最大发作评分3.38±1.94，IGF-1组为4.46±1.27，PPP组为2.53±2.0；大鼠急性期死亡率为IGF-1组30.77（4/13），对照组7.69%（1/13），PPP组没有大鼠死亡，IGF-1组和PPP组比较有显著统计学差异（P＜0.05）。PTZ模型对照组GTCS发病潜伏期均值为107.5±5.04(s)，IGF-1组为86.9±6.08(s)，PPP组为124.17±13.21(s)，IGF-1+PPP组为116.63±11.44(s)，与对照组比较，IGF-1组潜伏期明显缩短，PPP组潜伏期明显延长（P＜0.05）。GTCS发病比率IGF-1+PPP组为8/10，PPP组（6/10）与对照组（10/10）和IGF-1组（10/10）比较有显著统计学差异（P＜0.05）。PTZ模型IGF-1受体下游信号通路的活体干预研究显示，对照组GTCS发病潜伏期(s)均值为92.6±8.09，LY294002组为109.5±14.2，U0126组为105.83±8.75，LY294002+U0126组为122.5±16.67；与对照组比较，LY294002组和LY294002+U0126组潜伏期均明显延长，LY294002+U0126组与LY294002组和U0126组比较潜伏期也显著延长（P＜0.05）。GTCS发病比率对照组为10/10，LY294002组为8/10、U0126组和LY294002+U0126组均为6/10； U0126组和LY294002+U0126组与对照组比较均有显著统计学差异（P＜0.05）。结论：IGF-1增加PILO诱导SE发作比率，缩短潜伏期，增加最大发作评分和死亡率，缩短PTZ模型GTCS潜伏期，说明IGF-1增加可能有增加癫痫发作敏感性和SE严重程度的作用。PPP延长PILO诱导SE潜伏期，减少SE发作比率，降低SE评分和急性期大鼠死亡率；PPP，U0126和LY294002在延长PTZ模型GTCS潜伏期，减少GTCS发作比率方面都有一定效果；说明抑制IGF-1受体和下游信号通路可能有抑制癫痫活动的效果。第三部分IGF-1及其受体信号通路在癫痫发作中的细胞与分子机制目的：通过离体海马脑片IGF-1和PPP干预行膜片钳技术研究二者对癫痫模型海马兴奋性的影响，应用分子生物学技术检测IGF-1受体及下游信号活化状态，探讨IGF-1受体及其下游信号通路参与癫痫活动的分子机制。方法：用出生后14～16天乳鼠构建离体海马脑片，无镁诱导癫痫模型（对照组），IGF-1和PPP干预组（各组N=8-12），均记录场电位（fEPSP）轨迹，分析fEPSP斜率，记录无镁诱导痫样放电频率。观察微小兴奋性突触后电流（mEPSC）和兴奋性突触后电流（EPSC），包括：AMPAR和NMDAR介导的EPSC。IGF-1组在无镁人工脑脊液及记录液中加入IGF-1100ng/ml30min后记录，PPP组无镁诱导前加PPP5μM孵育1-2h，再给予无镁诱导，同时加IGF-1处理。用Western blot技术检测LiCL-PILO模型，正常对照、SE后6h组、24h组、7d组、21d组和60d组海马pIGF-1R/IGF-1R、pERK1/2/ERK1/2及pAKT/AKT蛋白水平的变化；检测IGF-1和PPP干预后6h和24h pIGF-1R/IGF-1RpERK1/2/ERK1/2及pAKT/AKT蛋白水平的变化。结果：IGF-1诱导大鼠海马脑片fEPSP斜率增加，增加无镁诱导痫样放电频率（P＜0.05）；PPP预处理，降低IGF-1诱导的fEPSP斜率增加，并减少无镁诱导痫样放电频率（P＜0.05）。与对照组比较，IGF-1增加mEPSC频率，PPP降低IGF-1诱导的mEPSC频率增加。mEPSC振幅累积分布曲线显示：和对照组组比较IGF-1组大鼠海马脑片mEPSC振幅累积分布曲线有显著偏移现象。IGF-1增加AMPAR和NMDAR介导的兴奋性突触后电流幅值，PPP可部分反转IGF-1诱导的EPSC的幅值。与正常对照组比较，PILO模型SE后急性期IGF-1R、ERK1/2及AKT磷酸化水平增加（P＜0.05）；IGF-1干预进一步增加SE后6h和24hIGF-1R、ERK1/2及AKT磷酸化水平（P＜0.05），而PPP则有抑制作用（P＜0.05）。结论：IGF-1诱导大鼠海马脑片兴奋性增加，PPP对IGF-1效应有一定的逆转作用，说明IGF-1及其受体通路可能通过兴奋性突触后电位起作用。IGF-1及PPP影响癫痫活动，可能通过IGF-1R及其下游ERK1/2及AKT信号通路的激活来实现的。第四部分miRNA-9调控IGF-1可能是抗癫痫发作新的治疗方法目的：课题组前期miRNAs芯片表达谱研究发现TLE患者颞叶皮质与对照组比较miRNA-9存在差异表达，靶基因预测IGF-1是miRNA-9的靶基因。实验检测TLE与对照组患者颞叶皮质和PILO模型miRNA-9表达，并验证二者的靶标关系。方法：用测定IGF-1mRNA同批RNA，用qRT-PCR技术检测miRNA-9表达水平。与广州锐博生物有限公司合作，采用双荧光素酶报告系统验证靶基因。结果：TLE患者颞叶皮质miRNA-9表达较对照组降低，但无显著统计学差异（P>0.05）。PILO模型miRNA-9在SE后6h和7d组较对照组表达显著增高（P<0.05），其它时间点表达差异不模型；双荧光素酶报告系统报告IGF-1是miRNA-9的靶基因。结论：miRNA-9和IGF-1表达在一定程度上有反向关系，结合双荧光素酶报告系统报告IGF-1是miRNA-9的靶基因，我们推测IGF-1可能是miRNA-9靶标，找到调控IGF-1基因新方法，通过miRNA-9调控IGF-1表达可能是一种新的抗癫痫靶标。更多还原

【Abstract】 PART ONE EXPRESSION OF IGF-1IN PATIENTS WITHTEMPORAL LOBE EPILEPSY AND EXPERIMENTAL RATSObjective To investigate the expression pattern of IGF-1in temporallobe tissue of intractable epilepsy patients and hippocampal tissue oflithium chloride-pilocarpine and pentamethazol induced rat models.Methods To detect the IGF-1protein and mRNA expression,24patients undergoing surgery for medically intractable TLE and12nonepileptic control subjects were examined by ELISA and quantitativereal-time PCR. Using the same technology tested the expression pattern ofIGF-1in temporal lobe tissue of experimental rats. Adult male SD ratsrandomly divided into normal control group of animals (n=5) and PILOinduced epilepsy groups (SE6h group,24h group,7d group,21d group,60d group, every group n=5), and PTZ induced epilepsy groups (dividedinto6h group and24h group, n=5).Results The expression of IGF-1protein and mRNA was increased intemporal neocortical of TLE patients (P<0.05). The protein expression of IGF-1was increased after SE6h and24h in pilocarpine and pentamethazoltreatment rats (P<0.05). The mRNA expression of IGF-1was increased inSE24h group,21d group and60d group of pilocarpine-induced rat models(P<0.05).Conclusion IGF-1expression is a significant increase in patients withtemporal lobe epilepsy and TLE rat model, therefore, our results indicatethat IGF-1may be involved in epileptogenesis and development ofepilepsy.PART TWO NEW DISCOVERIES OF EPILEPSY RESEARCH:IGF-1AND ITS RECEPTOR SIGNALING PATHWAYSREGULATED THE EPILEPTIC ACTIVITYObjective Rats by intracerebroventricular injection of recombinanthuman insulin-like growth factor (rhIGF-1) in vivo, and intraperitonealinjection of specific IGF-1receptor inhibitor (picropodophyllin, PPP) toobserve the effects on epilepsy rat models of PTZ and PILO inducedseizure activities. Rats by rat IGF-1receptor downstream signalingpathway MAPK-ERK inhibitors U0126and PI3K-AKT inhibitorLY294002in vivo, respectively, to observe effects on epilepsy rat modelsof PTZ activities.Methods All animals after anesthesia via Stereotactic embedded micro-injection catheter into the lateral ventricle. The rats were recoveredone week postoperative. Animals were observed the behavior change underthe waking state. Group: the control group (n=13), lateral ventricleinjection saline10μl; IGF-1Group (n=13), intracerebroventricular injectionrhIGF-110μg; PPP Group (n=15), intracerebroventricular injection NS10μl, intraperitoneal injection PPP20mg/kg weight; After intervention1hrespectively intraperitoneal injection PILO induced SE. PTZ model usedsame intervention method (n=10). LiCL-PILO model observed the latencytime of SE, Racine score and percentage SE. PTZ model observed thelatency time of SE and percentage SE. Both groups have recorded brainelectrical activity (n=3). U0126group (n=10), lateral ventricle injection ofU01265mM; LY294002group (n=10), LY29400220μM injected into thelateral ventricle; U0126+LY294002group (n=10), the U01265μM andLY29400220μM injected into the lateral ventricle. After intervention1hintraperitoneal injection of PTZ induced SE and observed the latency timeof SE and percentage SE.Results IGF-1shortened the latency LiCL-PILO and PTZ induced SEcompaired with the control group(P<0.05) and increased the4level aboveSE attack ratio and the severity of SE; PPP group, the incubation period ofthe onset is more lasting (P<0.05) and the ratio of4level above SE wassignificantly declined compaired with IGF-1Group. The serious degree ofSE was lightened in PPP group. The discharge frequency of IGF-1group is more against the control by the EEG observation and the PPP group wasobviously decreased. IGF-1shorten the incubation period compared withcontrol groups in PTZ kindling model (P<0.05), while the PPP Group wassignificantly extended in latency and reduced the spike discharge (P<0.05).LY294002was significantly extended incubation period in PTZ kindlingmodle than control groups (P<0.05), the ratio of GTCS was reduced, but nosignificant statistical differences (P>0.05). U0126was significantlyextended incubation period and reduced the ratio of GTCS in PTZ kindlingmodle compaired with control groups (P<0.05).Conclusion IGF-1increased the sensitivity of LiCL-PILO and PTZinduced seizure, while IGF-1receptor inhibitors PPP suppress seizureactivity.PART THREE THE CELLULAR AND MOLECULARMECHANISMS OF IGF-1AND ITS RECEPTOR SIGNALINGPATHWAYS IN EPILEPSYObjective To investigate the cellular and molecular mechanisms ofIGF-1and and its receptor signaling pathways in epilepsy. The patch-clamptechniques and western-bolt were used to investigate the role of IGF-1andPPP in epileptogenesis and the potential antiepilepsy mechanism throughthe intervention method in vivo and in vitro experiment study. Methods To evaluated weather the IGF-1and PPP can modifying theepileptic seizure and epileptiform discharge of rat models throughactivation of ERK and AKT signaling pathways, The patch-clamptechniques were used to detect the slope of field excitatory postsynapticpotential (fEPSP) and magnesium-free-induced the frequency of dischargein pilocarpine rats. Postnatal14-16days rats hippocampal slices, epilepsymodel induced by magnesium-free treatment (control group), theobservation of the effect of IGF-1and PPP on the amplitude and frequencyof miniature excitatory postsynaptic currents (mEPSC) in CA1pyramidalcells. We observed the effect of IGF-1and PPP on the evoked excitatorypostsynaptic currents (eEPSC, including NMDAR-mediated EPSC andAMPAR-mediated EPSC). Laboratory animals are divided into controlgroup, IGF-1(100ng/ml) group and PPP (5μM) add IGF-1(100ng/ml)Group (group n=12). Western blot detected the change of pIGF-1R/IGF-1R,pERK1/2/ERK1/2and pAKT/AKT protein level at control group, SE6hgroup,24h group,7d group,21d group and60d group in hippocampus ofpilocarpine rats. We investigated pIGF-1R/IGF-1R, pERK1/2/ERK1/2andpAKT/AKT protein expression at6h and24h after IGF-1and PPPintervention epileptic rats.Results The expression of pIGF-1R was increased at6h,24h,21d and60d group in hippocampus compaired with the control group in pilocarpinerats and pERK1/2and pAKT protein level was increased at6h and24h after SE(P<0.05). IGF-1increased the fEPSP the slope in hippocampalbrain slices of rats and the frequency of epileptiform discharges and thedischarge time induced by magnesium-free (P<0.05); Compared with thecontrol group, IGF-1increased mEPSC frequency while PPP decreased thefEPSP the slope and less mEPSC frequently. Cumulative distribution curveof amplitude display: IGF-1set of CA1area in the rat hippocampal slicemEPSC cumulative distribution curve amplitude apparent offset comparedwith the control group. IGF-1added NMDAR and AMPAR-mediatedexcitatory postsynaptic current amplitude while PPP lower IGF andmagnesium-free treatment in hippocampal slice neurons NMDAR andAMPAR-mediated EPSC amplitude.Conclusion IGF-1increased excitability of magnesium-freehippocampal slices in rats, but, PPP reduced the effection the IGF-1promotived-excitability of magnesium-free induced hippocampal slices.IGF-1and PPP works through NMDAR and AMPAR. IGF-1and PPPinfluence seizure activity which accomplished through the activation ofIGF-1R and their downstream ERK1/2and AKT signaling pathway. PART FOUR MIRNA-9REGULATION OF IGF-1GENEEXPRESSION MAY BE THE NEW ANTIEPILEPTIC TREATMENTObjective Our previous miRNAs microarray expression profilingstudies have found miRNA-9differences expression in patients with TLEtemporal cortical compared with the control group. The target geneprediction found that IGF-1is miRNA-9target gene. This study testmiRNA-9expression of temporal lobe cortex in patients with TLE and thecontrol group, and verify that the target of both relationships.Methods Using qRT-PCR technology determinated the expressionlevel of miRNA-9in patients with TLE and the control group. Wecooperated with Guangzhou Ribo biotechnology and verified target geneusing dual-luciferase report gene system.Results miRNA-9expression of temporal lobe cortex was reduced inpatients with TLE compared with a control group, but no significantstatistical differences (p＞0.05). miRNA-9expression increasedsignificantly at6h and7d after the SE compared with the control group inPILO model rats (P<0.05), and the expression was not significantlydifferences at other time point after SE. Dual-luciferase system reports thatIGF-1is the target genes of miRNA-9.Conclusion We infered that IGF-1is miRNA-9target and found thenew method for regulation of IGF-1gene. miRNA-9regulation of IGF-1 expression may be the new antiepileptic target.更多还原