本文選題：Dock3 + 癲癇形成　； 參考：《重慶醫(yī)科大學(xué)》2014年博士論文

【摘要】：第一部分：Dock3在難治性顳葉癲癇患者及匹羅卡品動物模型腦組織中的表達(dá) 目的： 腦組織中異常突觸傳遞及神經(jīng)網(wǎng)絡(luò)形成會影響神經(jīng)元興奮性，從而導(dǎo)致癲癇的產(chǎn)生。Dock3（dedicator of cytokinesis3）是一類新型鳥嘌呤核苷酸交換因子家族成員之一，能特異性激活Rho GTP酶中的Rac1，在中樞神經(jīng)系統(tǒng)及生長錐中聚集。通過調(diào)節(jié)神經(jīng)突觸的形成及軸突的定向生長，與神經(jīng)突觸傳遞及異常神經(jīng)網(wǎng)絡(luò)的形成有關(guān)。本研究首先檢測了Dock3在耐藥性顳葉癲癇（TLE）患者及氯化鋰-匹羅卡品癲癇動物模型中的表達(dá)情況，以探討Dock3在耐藥性顳葉癲癇發(fā)病中的作用。 方法： 1.將SD成年雄性大鼠隨機(jī)分為對照組（n=5只）和癲癇組（n=35只），癲癇組給予氯化鋰-匹羅卡品（LiCl-PILO）腹腔注射造模。其中癲癇組再分為7個不同時間點(diǎn)亞組（每組n=5只），即匹羅卡品誘導(dǎo)的癲癇持續(xù)狀態(tài)（SE）后6h組、1d組、3d組、1w組、2w組、1m組和2m組。其中1m組和2m組為慢性期（伴有自發(fā)性發(fā)作）。 2.從課題組所建立的癲癇腦組織標(biāo)本庫中隨機(jī)抽取24例耐藥性TLE患者和12例對照組患者的顳葉皮質(zhì)。 3.用免疫印跡和（或）免疫組化、qRT-PCR方法分別檢測Dock3在耐藥性TLE患者和（或）癲癇動物模型腦組織中的表達(dá)情況。 結(jié)果： 1.免疫組化染色結(jié)果顯示，Dock3染色陽性細(xì)胞呈棕黃色，主要在耐藥性TLE患者、對照組顳葉皮質(zhì)及匹羅卡品癲癇動物腦組織中神經(jīng)元的細(xì)胞膜和胞漿中表達(dá)。大鼠皮質(zhì)及海馬各區(qū)均有陽性細(xì)胞分布。其中在海馬齒狀回、CA3、CA1區(qū)陽性染色最強(qiáng)。統(tǒng)計(jì)學(xué)分析顯示耐藥性TLE患者與對照組相比平均光密度值明顯增高（p 0.05）。在大鼠皮質(zhì)及海馬組織中，Dock3的免疫組化平均OD值在匹羅卡品造模后與對照組相比差異明顯（p 0.05）。 2.免疫印跡分析顯示Dock3在耐藥性TLE患者顳葉皮質(zhì)及癲癇動物模型中表達(dá)水平顯著高于對照組（p 0.05）。匹羅卡品造模后各時間點(diǎn)相比差異不明顯（p0.05）。 3.免疫熒光染色結(jié)果顯示Dock3在耐藥性TLE患者顳葉皮質(zhì)及大鼠癲癇模型腦組織中主要與神經(jīng)元共表達(dá)，沒有與膠質(zhì)細(xì)胞共表達(dá)。 結(jié)論： Dock3在耐藥性TLE患者和匹羅卡品癲癇大鼠腦組織中表達(dá)均增高，提示其可能參與了耐藥性TLE的發(fā)病過程。 第二部分：Dock3shRNA沉默Dock3對癲癇行為學(xué)的影響 目的： 為進(jìn)一步明確Dock3是否會對癲癇發(fā)作及形成產(chǎn)生影響，我們在匹羅卡品癲癇模型及戊四氮點(diǎn)燃模型中利用Dock3基因的短發(fā)卡RNA（shRNA）慢病毒減少動物海馬內(nèi)源性Dock3表達(dá)，然后給予匹羅卡品和戊四氮造模并觀察對癲癇行為學(xué)以及對苔蘚纖維芽生的影響。 方法： 1. Dock3shRNA慢病毒采用海馬立體定位注射。C57/BL6小鼠在造模前分為三組：即立體定位雙側(cè)海馬注射生理鹽水組（Con），立體定位雙側(cè)海馬注射慢病毒空載體組(Vehicle shRNA)和立體定位雙側(cè)海馬注射Dock3shRNA慢病毒組(Dock3shRNA)。 2.在海馬立體定位注射Dock3shRNA慢病毒后，應(yīng)用激光共聚焦顯微鏡觀察轉(zhuǎn)染情況并用免疫印跡方法檢測其干預(yù)效率。 3.戊四氮點(diǎn)燃模型在每天給予腹腔注射亞劑量PTZ（35mg/Kg）后一個小時內(nèi)觀察發(fā)作的級別，當(dāng)腹腔注射PTZ后如果連續(xù)4天均出現(xiàn)了4級或以上級別發(fā)作，，為完全點(diǎn)燃。匹羅卡品模型在造模后1小時內(nèi)觀察癲癇行為學(xué)改變。每隔5分鐘記錄該時間段內(nèi)最高Racine評分及每只動物從腹腔注射匹羅卡品到出現(xiàn)第一次4級或4級以上發(fā)作所需時間。在癲癇的慢性期通過視頻記錄觀察在其慢性期自發(fā)發(fā)作（4級或4級以上）的次數(shù)。 4.應(yīng)用Timm染色對Dock3shRNA組、空病毒注射組和對照組進(jìn)行形態(tài)學(xué)觀察，以探討Dock3在癲癇異常網(wǎng)絡(luò)形成中的可能作用。 結(jié)果： 1. Dock3shRNA慢病毒海馬注射后，內(nèi)源性Dock3表達(dá)下降。在海馬注射后第5天和第6周，免疫印跡檢測與對照組相比，Dock3的表達(dá)在慢病毒注射后顯著下降（p0.05）。免疫熒光顯示慢病毒主要在在海馬、胼胝體部位表達(dá)，尤其是海馬CA3、CA1及齒狀回區(qū)。 2. Dock3shRNA海馬注射后，PTZ點(diǎn)燃模型中，Dock3shRNA組小鼠發(fā)作級別評分在第4、7、8、10、11和14天與對照組和空病毒組相比有明顯差異（p＜0.05）。Dock3shRNA組完全點(diǎn)燃所需時間為17.33±0.56（天），與對照組13.5±0.62（天）和空病毒組14.67±0.81（天）相比明顯延長（p＜0.05）。 3在匹羅卡品癲癇模型中，Dock3shRNA海馬注射后，急性期發(fā)作級別明顯降低，并且其潛伏期延長（p0.05）。慢性期內(nèi)觀察其自發(fā)發(fā)作頻率較空病毒組及對照組明顯減少（p0.05）。 4Dock3shRNA組Timm染色評分與空病毒組和對照組相比明顯減低（p0.05）。而空病毒組和對照組之間相比差別沒有統(tǒng)計(jì)學(xué)意義（p0.05）。 結(jié)論： 1. Dock3shRNA慢病毒干擾在第5天和動物行為學(xué)觀察完后的第6周均能有效降低內(nèi)源性Dock3表達(dá)。 2. Dock3shRNA慢病毒干擾能減輕急性期癲癇發(fā)作及慢性期自發(fā)發(fā)作程度。 3. Dock3對苔蘚纖維芽生有一定的作用，抑制Dock3能夠減低苔蘚纖維芽生的程度。 第三部分Dock3在大鼠癲癇發(fā)作中的細(xì)胞機(jī)制 目的： 為了探討Dock3對癲癇發(fā)作影響的作用機(jī)制，我們應(yīng)用全細(xì)胞膜片鉗技術(shù)對Dock3shRNA干預(yù)的腦片進(jìn)行電生理研究，觀察其對癇性放電的影響。 方法： 1.將實(shí)驗(yàn)動物隨機(jī)分為三組：海馬注射生理鹽水組（control）、空載體組(Vehicle shRNA.)、Dock3shRNA組(Dock3shRNA)。小鼠腦片應(yīng)用無鎂人工腦脊液誘發(fā)的癲癇細(xì)胞模型進(jìn)行電生理觀察。 2.用全細(xì)胞膜片鉗電生理技術(shù)分別記錄三組海馬CA1區(qū)神經(jīng)元細(xì)胞的動作電位（AP）、微小興奮性突觸后電流（mEPSC）以及誘發(fā)的興奮性突觸后電流（eEPSC）。比較各組AP的放電頻率、NMDA/AMPA受體幅值比以及mEPSC和eEPSC的幅值和頻率。 結(jié)果： 1. Dock3shRNA組神經(jīng)元與對照組和空病毒組相比，自發(fā)性動作電位的頻率明顯減低，統(tǒng)計(jì)學(xué)分析有顯著差異（p 0.05）。對照組和空病毒組相比，差異無統(tǒng)計(jì)學(xué)意義（p0.05）。 2. Dock3shRNA對mEPSC的影響：（1）對頻率的影響：Dock3shRNA組神經(jīng)元mEPSC頻率明顯降低。其差異有統(tǒng)計(jì)學(xué)意義(p 0.05)。對照組和空病毒組兩者相比，差別無顯著性（p0.05）。（2）對波幅的影響：與對照組及空病毒組相比，Dock3shRNA組神經(jīng)元mEPSC幅值無明顯改變，差別無顯著性（p0.05）。 3. Dock3shRNA組NMDA/AMPA的幅值比與對照組和空病毒組相比明顯減低(p 0.05)。NMDA受體介導(dǎo)的EPSCs幅值與對照組和空病毒組相比明顯降低(p 0.05)。而AMPA受體介導(dǎo)的EPSCs幅值則沒有改變(p0.05)。 結(jié)論： 1. Dock3shRNA能有效抑制海馬神經(jīng)元的自發(fā)性放電頻率。減輕神經(jīng)元的興奮性。 2. Dock3shRNA能夠抑制海馬腦片中錐體神經(jīng)元的mEPSC的頻率改變。 3. Dock3shRNA能夠有效抑制海馬腦片NMDA受體介導(dǎo)的突觸后電流幅值，而對AMPA介導(dǎo)的突觸電流沒有影響。 第四部分Rac1在Dock3對癲癇的影響中的作用 目的： 觀察Rac1在癲癇患者及動物模型中的表達(dá)，為了探討Rac1在Dock3對癲癇影響中的可能作用，我們應(yīng)用Rac1活性抑制劑NSC23766將Rac1活性抑制后，再用Dock3慢病毒進(jìn)行干預(yù)，觀察Dock3shRNA是否還對癲癇行為學(xué)及形態(tài)學(xué)有影響。 方法： 1. Rac1抑制劑注射采用側(cè)腦室置管方法連續(xù)給藥兩周，共分為三組（NSC23766兩個劑量）：即側(cè)腦室注射生理鹽水組（Con），側(cè)腦室注射NSC23766組(NSC2376650μM)和側(cè)腦室注射NSC23766組(NSC23766100μM)。Dock3shRNA與NSC23766聯(lián)合用藥分組：根據(jù)所注射試劑的不同分為三組：生理鹽水+NSC23766組（control）、空載體組+NSC23766組(Vehicle shRNA.)、Dock3shRNA+NSC23766組(Dock3shRNA)。 2.行為學(xué)觀察：戊四氮點(diǎn)燃模型在每天腹腔注射亞劑量PTZ（35mg/Kg）后一個小時內(nèi)觀察發(fā)作的最高級別，腹腔注射PTZ后如果連續(xù)4天均出現(xiàn)4級或以上級別發(fā)作，為完全點(diǎn)燃。匹羅卡品模型在造模后50min之內(nèi)觀察癲癇行為學(xué)改變。每隔5分鐘記錄該時間段內(nèi)最高Racine評分及每只動物從腹腔注射匹羅卡品到出現(xiàn)第一次4級或4級以上發(fā)作所需時間。在癲癇的慢性期通過視頻記錄觀察在其慢性期自發(fā)發(fā)作（4級或4級以上）的次數(shù)。 3.在Dock3shRNA干預(yù)時聯(lián)合應(yīng)用NSC23766，觀察完慢性期行為學(xué)改變及苔蘚纖維芽生情況后，用免疫印跡實(shí)驗(yàn)檢測Dock3和Rac1活性。 結(jié)果： 1.免疫印跡分析顯示，與對照組相比，不論是Rac1還是其活性形式Rac1-GTP其表達(dá)都要高于對照組（p0.05）。應(yīng)用Rac1抑制劑NSC23766側(cè)腦室注射后，免疫印跡檢測Rac1-GTP表達(dá)量與對照組相比明顯降低(p 0.05)。 2.應(yīng)用NSC23766后，在匹羅卡品癲癇模型中，急性期發(fā)作級別明顯降低，并且其潛伏期延長。在PTZ點(diǎn)燃模型中，NSC23766組發(fā)作級別在第5、6、8-10、13-15和17天與對照組相比有明顯差異，完全點(diǎn)燃所需時間明顯延長（p0.05）。 3.當(dāng)聯(lián)合應(yīng)用Dock3shRNA和NSC23766將Rac1活性抑制后，在匹羅卡品癲癇模型中，Dock3shRNA組與空病毒組及對照組相比，急性期顯示發(fā)作級別無差別（p＞0.05），慢性期觀察自發(fā)發(fā)作次數(shù)未看到各組之間有差異（p0.05）。Dock3shRNA組Timm染色評分與空病毒注射和對照組相比，差別無統(tǒng)計(jì)學(xué)意義（p0.05）。 結(jié)論： 1.免疫印跡分析顯示Rac1/Rac1-GTP在耐藥性TLE患者顳葉皮質(zhì)及癲癇動物模型中表達(dá)水平顯著高于對照組。 2.在匹羅卡品癲癇模型和PTZ點(diǎn)燃模型中，應(yīng)用NSC23766均能減輕其癲癇發(fā)作的程度。 3.當(dāng)Rac1抑制劑NSC23766應(yīng)用后，Dock3shRNA對癲癇行為學(xué)的及苔蘚纖維芽生的影響消失，說明Rac1在Dock3對癲癇的影響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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R742.1

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相關(guān)期刊論文 前1條

1 肖飛;何梅;王學(xué)峰;席志芹;李勁梅;吳原;鐘正福;朱丹;盧涌;李紅衛(wèi);;耐藥性顳葉癲癇患者腦組織中Cdc42的表達(dá)[J];中華醫(yī)學(xué)雜志;2007年29期

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