Dock3在癫痫发作中的作用及其可能的机制研究

发布时间：2018-07-04 06:45

本文选题：Dock3 + 癫痫形成　；参考：《重庆医科大学》2014年博士论文

【摘要】：第一部分：Dock3在难治性颞叶癫痫患者及匹罗卡品动物模型脑组织中的表达目的：脑组织中异常突触传递及神经网络形成会影响神经元兴奋性，从而导致癫痫的产生。Dock3（dedicator of cytokinesis3）是一类新型鸟嘌呤核苷酸交换因子家族成员之一，能特异性激活Rho GTP酶中的Rac1，在中枢神经系统及生长锥中聚集。通过调节神经突触的形成及轴突的定向生长，与神经突触传递及异常神经网络的形成有关。本研究首先检测了Dock3在耐药性颞叶癫痫（TLE）患者及氯化锂-匹罗卡品癫痫动物模型中的表达情况，以探讨Dock3在耐药性颞叶癫痫发病中的作用。方法： 1.将SD成年雄性大鼠随机分为对照组（n=5只）和癫痫组（n=35只），癫痫组给予氯化锂-匹罗卡品（LiCl-PILO）腹腔注射造模。其中癫痫组再分为7个不同时间点亚组（每组n=5只），即匹罗卡品诱导的癫痫持续状态（SE）后6h组、1d组、3d组、1w组、2w组、1m组和2m组。其中1m组和2m组为慢性期（伴有自发性发作）。 2.从课题组所建立的癫痫脑组织标本库中随机抽取24例耐药性TLE患者和12例对照组患者的颞叶皮质。 3.用免疫印迹和（或）免疫组化、qRT-PCR方法分别检测Dock3在耐药性TLE患者和（或）癫痫动物模型脑组织中的表达情况。结果： 1.免疫组化染色结果显示，Dock3染色阳性细胞呈棕黄色，主要在耐药性TLE患者、对照组颞叶皮质及匹罗卡品癫痫动物脑组织中神经元的细胞膜和胞浆中表达。大鼠皮质及海马各区均有阳性细胞分布。其中在海马齿状回、CA3、CA1区阳性染色最强。统计学分析显示耐药性TLE患者与对照组相比平均光密度值明显增高（p 0.05）。在大鼠皮质及海马组织中，Dock3的免疫组化平均OD值在匹罗卡品造模后与对照组相比差异明显（p 0.05）。 2.免疫印迹分析显示Dock3在耐药性TLE患者颞叶皮质及癫痫动物模型中表达水平显著高于对照组（p 0.05）。匹罗卡品造模后各时间点相比差异不明显（p0.05）。 3.免疫荧光染色结果显示Dock3在耐药性TLE患者颞叶皮质及大鼠癫痫模型脑组织中主要与神经元共表达，没有与胶质细胞共表达。结论： Dock3在耐药性TLE患者和匹罗卡品癫痫大鼠脑组织中表达均增高，提示其可能参与了耐药性TLE的发病过程。第二部分：Dock3shRNA沉默Dock3对癫痫行为学的影响目的：为进一步明确Dock3是否会对癫痫发作及形成产生影响，我们在匹罗卡品癫痫模型及戊四氮点燃模型中利用Dock3基因的短发卡RNA（shRNA）慢病毒减少动物海马内源性Dock3表达，然后给予匹罗卡品和戊四氮造模并观察对癫痫行为学以及对苔藓纤维芽生的影响。方法： 1. Dock3shRNA慢病毒采用海马立体定位注射。C57/BL6小鼠在造模前分为三组：即立体定位双侧海马注射生理盐水组（Con），立体定位双侧海马注射慢病毒空载体组(Vehicle shRNA)和立体定位双侧海马注射Dock3shRNA慢病毒组(Dock3shRNA)。 2.在海马立体定位注射Dock3shRNA慢病毒后，应用激光共聚焦显微镜观察转染情况并用免疫印迹方法检测其干预效率。 3.戊四氮点燃模型在每天给予腹腔注射亚剂量PTZ（35mg/Kg）后一个小时内观察发作的级别，当腹腔注射PTZ后如果连续4天均出现了4级或以上级别发作，，为完全点燃。匹罗卡品模型在造模后1小时内观察癫痫行为学改变。每隔5分钟记录该时间段内最高Racine评分及每只动物从腹腔注射匹罗卡品到出现第一次4级或4级以上发作所需时间。在癫痫的慢性期通过视频记录观察在其慢性期自发发作（4级或4级以上）的次数。 4.应用Timm染色对Dock3shRNA组、空病毒注射组和对照组进行形态学观察，以探讨Dock3在癫痫异常网络形成中的可能作用。结果： 1. Dock3shRNA慢病毒海马注射后，内源性Dock3表达下降。在海马注射后第5天和第6周，免疫印迹检测与对照组相比，Dock3的表达在慢病毒注射后显著下降（p0.05）。免疫荧光显示慢病毒主要在在海马、胼胝体部位表达，尤其是海马CA3、CA1及齿状回区。 2. Dock3shRNA海马注射后，PTZ点燃模型中，Dock3shRNA组小鼠发作级别评分在第4、7、8、10、11和14天与对照组和空病毒组相比有明显差异（p＜0.05）。Dock3shRNA组完全点燃所需时间为17.33±0.56（天），与对照组13.5±0.62（天）和空病毒组14.67±0.81（天）相比明显延长（p＜0.05）。 3在匹罗卡品癫痫模型中，Dock3shRNA海马注射后，急性期发作级别明显降低，并且其潜伏期延长（p0.05）。慢性期内观察其自发发作频率较空病毒组及对照组明显减少（p0.05）。 4Dock3shRNA组Timm染色评分与空病毒组和对照组相比明显减低（p0.05）。而空病毒组和对照组之间相比差别没有统计学意义（p0.05）。结论： 1. Dock3shRNA慢病毒干扰在第5天和动物行为学观察完后的第6周均能有效降低内源性Dock3表达。 2. Dock3shRNA慢病毒干扰能减轻急性期癫痫发作及慢性期自发发作程度。 3. Dock3对苔藓纤维芽生有一定的作用，抑制Dock3能够减低苔藓纤维芽生的程度。第三部分Dock3在大鼠癫痫发作中的细胞机制目的：为了探讨Dock3对癫痫发作影响的作用机制，我们应用全细胞膜片钳技术对Dock3shRNA干预的脑片进行电生理研究，观察其对痫性放电的影响。方法： 1.将实验动物随机分为三组：海马注射生理盐水组（control）、空载体组(Vehicle shRNA.)、Dock3shRNA组(Dock3shRNA)。小鼠脑片应用无镁人工脑脊液诱发的癫痫细胞模型进行电生理观察。 2.用全细胞膜片钳电生理技术分别记录三组海马CA1区神经元细胞的动作电位（AP）、微小兴奋性突触后电流（mEPSC）以及诱发的兴奋性突触后电流（eEPSC）。比较各组AP的放电频率、NMDA/AMPA受体幅值比以及mEPSC和eEPSC的幅值和频率。结果： 1. Dock3shRNA组神经元与对照组和空病毒组相比，自发性动作电位的频率明显减低，统计学分析有显著差异（p 0.05）。对照组和空病毒组相比，差异无统计学意义（p0.05）。 2. Dock3shRNA对mEPSC的影响：（1）对频率的影响：Dock3shRNA组神经元mEPSC频率明显降低。其差异有统计学意义(p 0.05)。对照组和空病毒组两者相比，差别无显著性（p0.05）。（2）对波幅的影响：与对照组及空病毒组相比，Dock3shRNA组神经元mEPSC幅值无明显改变，差别无显著性（p0.05）。 3. Dock3shRNA组NMDA/AMPA的幅值比与对照组和空病毒组相比明显减低(p 0.05)。NMDA受体介导的EPSCs幅值与对照组和空病毒组相比明显降低(p 0.05)。而AMPA受体介导的EPSCs幅值则没有改变(p0.05)。结论： 1. Dock3shRNA能有效抑制海马神经元的自发性放电频率。减轻神经元的兴奋性。 2. Dock3shRNA能够抑制海马脑片中锥体神经元的mEPSC的频率改变。 3. Dock3shRNA能够有效抑制海马脑片NMDA受体介导的突触后电流幅值，而对AMPA介导的突触电流没有影响。第四部分Rac1在Dock3对癫痫的影响中的作用目的：观察Rac1在癫痫患者及动物模型中的表达，为了探讨Rac1在Dock3对癫痫影响中的可能作用，我们应用Rac1活性抑制剂NSC23766将Rac1活性抑制后，再用Dock3慢病毒进行干预，观察Dock3shRNA是否还对癫痫行为学及形态学有影响。方法： 1. Rac1抑制剂注射采用侧脑室置管方法连续给药两周，共分为三组（NSC23766两个剂量）：即侧脑室注射生理盐水组（Con），侧脑室注射NSC23766组(NSC2376650μM)和侧脑室注射NSC23766组(NSC23766100μM)。Dock3shRNA与NSC23766联合用药分组：根据所注射试剂的不同分为三组：生理盐水+NSC23766组（control）、空载体组+NSC23766组(Vehicle shRNA.)、Dock3shRNA+NSC23766组(Dock3shRNA)。 2.行为学观察：戊四氮点燃模型在每天腹腔注射亚剂量PTZ（35mg/Kg）后一个小时内观察发作的最高级别，腹腔注射PTZ后如果连续4天均出现4级或以上级别发作，为完全点燃。匹罗卡品模型在造模后50min之内观察癫痫行为学改变。每隔5分钟记录该时间段内最高Racine评分及每只动物从腹腔注射匹罗卡品到出现第一次4级或4级以上发作所需时间。在癫痫的慢性期通过视频记录观察在其慢性期自发发作（4级或4级以上）的次数。 3.在Dock3shRNA干预时联合应用NSC23766，观察完慢性期行为学改变及苔藓纤维芽生情况后，用免疫印迹实验检测Dock3和Rac1活性。结果： 1.免疫印迹分析显示，与对照组相比，不论是Rac1还是其活性形式Rac1-GTP其表达都要高于对照组（p0.05）。应用Rac1抑制剂NSC23766侧脑室注射后，免疫印迹检测Rac1-GTP表达量与对照组相比明显降低(p 0.05)。 2.应用NSC23766后，在匹罗卡品癫痫模型中，急性期发作级别明显降低，并且其潜伏期延长。在PTZ点燃模型中，NSC23766组发作级别在第5、6、8-10、13-15和17天与对照组相比有明显差异，完全点燃所需时间明显延长（p0.05）。 3.当联合应用Dock3shRNA和NSC23766将Rac1活性抑制后，在匹罗卡品癫痫模型中，Dock3shRNA组与空病毒组及对照组相比，急性期显示发作级别无差别（p＞0.05），慢性期观察自发发作次数未看到各组之间有差异（p0.05）。Dock3shRNA组Timm染色评分与空病毒注射和对照组相比，差别无统计学意义（p0.05）。结论： 1.免疫印迹分析显示Rac1/Rac1-GTP在耐药性TLE患者颞叶皮质及癫痫动物模型中表达水平显著高于对照组。 2.在匹罗卡品癫痫模型和PTZ点燃模型中，应用NSC23766均能减轻其癫痫发作的程度。 3.当Rac1抑制剂NSC23766应用后，Dock3shRNA对癫痫行为学的及苔藓纤维芽生的影响消失，说明Rac1在Dock3对癫痫的影响中可能起了重要作用。
[Abstract]:Part one: the expression of Dock3 in intractable temporal lobe epilepsy and pilocarpine animal models.
Objective:
Abnormal synaptic transmission and neural network formation in the brain can affect neuronal excitability, resulting in.Dock3 (dedicator of cytokinesis3), one of the new family members of a new class of guanine nucleotide exchange factors, which can specifically activate the Rac1 in the Rho GTP enzyme and accumulate in the central nervous system and the growth cone. The formation of synapses and the directional growth of axons are related to the transmission of synapses and the formation of abnormal neural networks. In this study, we first detected the expression of Dock3 in patients with drug-resistant temporal lobe epilepsy (TLE) and lithium pilocarpine, in order to explore the role of Dock3 in the pathogenesis of drug-resistant temporal lobe epilepsy.
Method:
1. SD adult male rats were randomly divided into control group (n=5) and epilepsy group (n=35 only), epilepsy group was given lithium chloride pilocarpine (LiCl-PILO) intraperitoneal injection molding. The epileptic group was divided into 7 different time point subgroups (each group n=5), that is, pilocarpine induced epileptic status (SE), 6h group, 1D group, 3D group, 1W group, 2W group, 1m group and group. Among them, group 1m and group 2m were chronic (with spontaneous seizures).
2. randomly selected 24 cases of drug resistant TLE patients and 12 cases of control group's temporal cortex from the epileptic brain tissue database set up by the research group.
3. Western blotting and / or immunohistochemistry and qRT-PCR were used to detect the expression of Dock3 in the brain tissues of drug resistant TLE and / or epileptic animal models.
Result:
1. the results of immunohistochemical staining showed that the positive cells of Dock3 staining were brown and yellow, mainly in the membrane and cytoplasm of neurons in the temporal cortex and pilocarpine epileptic animal brain tissue in the drug resistant TLE patients and the control group. The positive cells in the cortex and hippocampus of the rats were distributed in the hippocampus and the dentate gyrus, CA3 and CA1 region. Statistical analysis showed that the average optical density of the drug resistant TLE patients was significantly higher than that of the control group (P 0.05). In the cortex and hippocampus of rats, the average OD value of Dock3 was significantly different from the control group after pilocarpine model (P 0.05).
2. Western blot analysis showed that the expression level of Dock3 in the temporal lobe cortex and epileptic animal model of patients with drug-resistant TLE was significantly higher than that of the control group (P 0.05). The difference of the time points of pilocarpine model was not significant (P0.05).
3. the results of immunofluorescence staining showed that Dock3 was co expressed with neurons in the temporal lobe cortex and the rat model of epileptic model of the drug-resistant TLE patients, and did not co express glial cells.
Conclusion:
The expression of Dock3 increased in the brain tissues of drug resistant TLE patients and pilocarpine induced epilepsy rats, suggesting that it might be involved in the pathogenesis of drug-resistant TLE.
The second part: the effect of Dock3shRNA silencing Dock3 on epileptic behavior.
Objective:
To further determine whether Dock3 could affect epileptic seizures and formation, we used the pilocarpine epilepsy model and the amyl four nitrogen kindling model to reduce endogenous Dock3 expression in the hippocampus by using the Dock3 gene's short hair card RNA (shRNA) lentivirus, and then give pilocarpine and amyl four nitrogen models and observe the behavior of epilepsy and the moss. The effects of moss fiber on the sprout.
Method:
1. Dock3shRNA lentivirus was divided into three groups: stereotaxic hippocampus injection group (Con), stereotaxic hippocampal injection of lentivirus (Vehicle shRNA) and stereotaxic hippocampal injection of Dock3shRNA lentivirus group (Dock3shRNA).
2. after injection of Dock3shRNA lentivirus into hippocampus, the transfection rate was observed by laser scanning confocal microscope and the intervention efficiency was detected by Western blot.
The 3. amyl four nitrogen kindling model observed the level of the seizure within an hour after an intraperitoneal injection of PTZ (35mg/Kg) every day. After the intraperitoneal injection of PTZ, a class of 4 or more episodes were observed for 4 days. The pilocarpine model observed the changes in the epileptic behavior within 1 hours after the model. The time was recorded every 5 minutes. The highest Racine score in the segment and the time required for each animal from intraperitoneal injection of pilocarpine to the first stage of the first 4 or more than 4 levels. In the chronic period of epilepsy, the frequency of spontaneous episodes (4 or more than grade 4) in the chronic phase of the seizure was recorded.
4. Timm staining was used to observe the morphology of Dock3shRNA group, empty virus injection group and control group in order to explore the possible role of Dock3 in the formation of abnormal network of epilepsy.
Result:
The expression of endogenous Dock3 decreased after the injection of 1. Dock3shRNA lentivirus in the hippocampus. The expression of Dock3 was significantly decreased after the fifth and sixth weeks after the injection of the hippocampus, compared with the control group (P0.05). The immunofluorescence showed that the lentivirus was mainly expressed in the hippocampus and the corpus callosum, especially in the hippocampus CA3, CA1 and dentate gyrus.
2. Dock3shRNA after hippocampal injection, in the PTZ kindling model, the grade score of the Dock3shRNA group was significantly different from the control group and the air virus group on the 4,7,8,10,11 and the 14 days (P < 0.05) the time required for the complete kindling of the.Dock3shRNA group was 17.33 + 0.56 (days), compared with the control group, 13.5 + 0.62 (days) and the empty virus group 14.67 + 0.81 (day). Long (P < 0.05).
3 in the pilocarpine epilepsy model, after the injection of Dock3shRNA hippocampus, the acute stage of attack was significantly reduced and its incubation period was prolonged (P0.05). The spontaneous episodes of the seizures in the chronic period were significantly less than those in the air virus group and the control group (P0.05).
The score of Timm staining in group 4Dock3shRNA was significantly lower than that in the air virus group and the control group (P0.05), but there was no significant difference in the difference between the air virus group and the control group (P0.05).
Conclusion:
1. Dock3shRNA lentivirus interference can effectively reduce endogenous Dock3 expression in fifth days and sixth weeks after animal behavior observation.
2. Dock3shRNA lentivirus interference can reduce the incidence of acute seizures and spontaneous seizures in chronic phase.
3. Dock3 has a certain effect on the development of mossy fiber buds, and inhibition of Dock3 can reduce the degree of bryophyte budding.
The third part is the cellular mechanism of Dock3 in epileptic seizures in rats.
Objective:
In order to explore the mechanism of the effect of Dock3 on epileptic seizures, we applied the whole cell patch clamp technique to the electrophysiological study of the Dock3shRNA interfered brain slices to observe the effect on the epileptic discharge.
Method:
1. the experimental animals were randomly divided into three groups: hippocampal injection of saline group (control), no-load group (Vehicle shRNA.) and group Dock3shRNA (Dock3shRNA). The epileptic cell model induced by magnesium free cerebrospinal fluid (CSF) induced by magnesium free cerebrospinal fluid was observed by electrophysiological observation.
2. the action potential (AP), the minute excitatory post synaptic current (mEPSC) and the induced excitatory postsynaptic current (eEPSC) were recorded by the whole cell patch clamp electrophysiological technique. The discharge frequency of AP, the amplitude ratio of NMDA/AMPA receptor and the amplitude and frequency of mEPSC and eEPSC were compared in each group of hippocampal CA1 area neurons.
Result:
1. Dock3shRNA group neurons compared with the control group and the empty virus group, the frequency of spontaneous action potential decreased significantly, statistically significant difference (P 0.05). There was no significant difference between the control group and the air virus group (P0.05).
2. Dock3shRNA effect on mEPSC: (1) effect on frequency: the mEPSC frequency of Dock3shRNA group was significantly reduced. The difference was statistically significant (P 0.05). There was no significant difference between the control group and the empty virus group (P0.05). (2) the influence of the amplitude on the amplitude: the mEPSC amplitude of the Dock3shRNA group was not clear compared with the control group and the empty virus group. There was no significant difference in the difference (P0.05).
The amplitude of NMDA/AMPA in group 3. Dock3shRNA was significantly lower than that in the control group and the air virus group (P 0.05), the EPSCs amplitude of.NMDA receptor mediated by.NMDA receptor was significantly lower than that of the control group and the empty virus group (P 0.05), while the EPSCs amplitude mediated by AMPA receptor was not changed (P0.05).
Conclusion:
1. Dock3shRNA can effectively inhibit the spontaneous firing frequency of hippocampal neurons and alleviate the excitability of neurons.
2. Dock3shRNA could inhibit the change of mEPSC frequency in pyramidal neurons of hippocampal slices.
3. Dock3shRNA can effectively inhibit the amplitude of NMDA receptor mediated postsynaptic currents in hippocampal slices, but has no effect on AMPA mediated synaptic currents.
The fourth part is the role of Rac1 in the effect of Dock3 on epilepsy.
Objective:
To observe the expression of Rac1 in epileptic patients and animal models, in order to explore the possible role of Rac1 in the effect of Dock3 on epilepsy, we used the Rac1 activity inhibitor NSC23766 to inhibit the activity of Rac1, and then the Dock3 lentivirus was used to observe the effect of Dock3shRNA on the behavior and morphology of epilepsy.
Method:
1. Rac1 inhibitors were injected with the lateral ventricle catheterization for two weeks, which were divided into three groups (NSC23766 and two doses): the lateral ventricle injection of saline group (Con), the side ventricle injection NSC23766 group (NSC2376650 mu M) and the side ventricle injection NSC23766 group (NSC23766100 u M).Dock3shRNA and NSC23766 combined medication group: according to the injected reagent The difference was divided into three groups: saline +NSC23766 group (control), empty carrier group +NSC23766 group (Vehicle shRNA.), Dock3shRNA+NSC23766 group (Dock3shRNA).
2. behavioral observation: the amyl four nitrogen kindling model observed the highest level of the attack within an hour after intraperitoneal injection of PTZ (35mg/Kg) every day. After intraperitoneal injection of PTZ, a class of 4 or more episodes were observed for 4 days. The pilocarpine model observed the behavior changes within 50min after the model. Every 5 minutes was recorded. The highest Racine score within the time period and the time required for each animal from intraperitoneal injection of pilocarpine to the first stage 4 or more than 4 levels were recorded. In the chronic period of epilepsy, the frequency of spontaneous episodes (4 or more than grade 4) in the chronic phase of the seizure was recorded.
3. the activity of Dock3 and Rac1 were detected by immunoblotting after the combined application of NSC23766 during the intervention of Dock3shRNA. After observing the behavior changes of the chronic phase and the bryophyte buds, the immunoblotting test was used to detect the activity of Dock3 and Rac1.
Result:
1. Western blot analysis showed that compared with the control group, the expression of Rac1 and its active Rac1-GTP was higher than that of the control group (P0.05). Compared with the control group, the expression of Rac1-GTP was significantly lower than that of the control group (P 0.05) after the injection of Rac1 inhibitor NSC23766 side ventricle.
2. after the use of NSC23766, in the pilocarpine epilepsy model, the acute stage of attack was significantly reduced and its incubation period was prolonged. In the PTZ kindling model, the seizure level in group NSC23766 was significantly different from the control group at 5,6,8-10,13-15 and 17 days, and the time required for the complete kindling was significantly prolonged (P0.05).
3. when the Rac1 activity was suppressed by combined use of Dock3shRNA and NSC23766, in the pilocarpine epilepsy model, the acute stage showed no difference between the Dock3shRNA group and the empty virus group and the control group (P > 0.05). The frequency of spontaneous episodes in the chronic phase did not see the difference between each group (P0.05) the Timm staining score of the.Dock3shRNA group and the empty virus. The difference between injection and control group was not statistically significant (P0.05).
Conclusion:
1. Western blot analysis showed that the expression level of Rac1/Rac1-GTP in the temporal lobe cortex and epilepsy animal models of drug resistant TLE was significantly higher than that of the control group.
2. in the pilocarpine epilepsy model and PTZ kindling model, NSC23766 can reduce the degree of epileptic seizure.
3. after the application of Rac1 inhibitor NSC23766, the effect of Dock3shRNA on epilepsy and mossy fiber buds disappeared, suggesting that Rac1 may play an important role in the impact of Dock3 on epilepsy.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.1

【参考文献】