本文選題：腦炎 + GSK-3β　； 參考：《川北醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:通過側(cè)腦室注射脂多糖(LPS)構(gòu)建成年SD大鼠腦炎模型,并檢測海馬組織中的炎性因子及GSK-3β的表達變化。觀察不同劑量鋰鹽對該模型大鼠腦組織病理生理變化、GSK-3β表達及癇性發(fā)作敏感性的影響;應(yīng)用GSK-3β激動劑WT、其間接抑制劑VPA進一步干預(yù)模型大鼠,觀察GSK-3β在腦炎及腦炎后癲癇發(fā)作中的作用。以期初步探討鋰鹽對腦炎和腦炎后癲癇發(fā)作的影響及可能機制,并為其防治提供新思路。方法:(1)LPS顱內(nèi)感染大鼠模型的制備:將20只成年雄性SD大鼠隨機分為對照組和LPS組,每組10只,LPS組大鼠側(cè)腦室注射LPS50μg/5ul,對照組注射同等體積生理鹽水(NS)。給藥24小時后,每組隨機選取5只大鼠麻醉,斷頭取腦,分離雙側(cè)海馬組織,提取蛋白,采用ELISA檢測IL-1β及TNF-α;采用Western-blot方法檢測P-GSK-3β及GSK-3β。各組剩余5只大鼠灌注取腦,制作石蠟切片用作免疫組化檢測腦組織的病理變化。(2)不同劑量鋰鹽對LPS致炎和癲癇發(fā)作的影響:取48只成年雄性SD大鼠,隨機分為6組:對照組、LPS組、LPS-10mg Li組、LPS-20mgLi組、LPS-40mgLi組、LPS-80mgLi組,每組各8只,采用上述方法腦炎模型組側(cè)腦室注入LPS 50μg/5μl,對照組注射同等體積生理鹽水。3小時后腹腔分別注射5ml/kg的生理鹽水(NS)、NS、10、20、40、80mg/Kg的Licl,每24小時一次,連續(xù)3天。然后行腹腔注射匹魯卡品(Pilo),觀察各組大鼠癲癇行為學(xué)表現(xiàn)。(3)不同劑量鋰鹽對lps顱內(nèi)感染大鼠海馬病理生理與癲癇發(fā)作的影響及其可能機制:取126只成年雄性sd大鼠,隨機分為lps-10mgli組、lps-20mgli組、lps-40mgli組、lps-80mgli組、lps-vpa組、lps-wt組和lps組,每組18只,采用上述方法腦炎模型組側(cè)腦室注入lps50μg/5μl,3小時后腹腔分別注射10、20、40、80mg/kglicl、30mg/kgvpa、0.6μg/kgwt和5ml/kg生理鹽水,每24小時一次,連續(xù)3天。每組隨機選取8只大鼠腹腔注射pilo,行顱內(nèi)電極腦電圖觀察基礎(chǔ)腦電頻譜、能譜、發(fā)作后高頻振蕩活動變化及癲癇發(fā)作潛伏期與嚴重程度觀察;另外每組隨機選取5只大鼠麻醉斷頭取雙側(cè)海馬組織,提取蛋白,采用elisa檢測il-1β、tnf-α、il-10的濃度,western-blot檢測p-gsk-3β及gsk-3β的表達量;剩余的大鼠采用免疫組化檢測海馬神經(jīng)元和小膠質(zhì)細胞表達。結(jié)果:(1)與對照組相比:lps組大鼠體溫升高,進食、活動減少,且海馬組織中il-1β、tnf-α的濃度明顯升高(p0.05);lps組大鼠對pilo的致癇性增強,se潛伏期明顯縮短(p0.05),同時該組大鼠海馬組織中g(shù)sk-3β的含量較對照組明顯升高,而p-gsk-3β的含量較對照組明顯降低(p0.05);海馬ca1區(qū)有大量活化增生的小膠質(zhì)細胞,神經(jīng)元排列松散、紊亂、脫失、形態(tài)模糊,染色變淺,形態(tài)不完整,與對照組比較有顯著差異(p0.05)。(2)與lps組比較:小劑量鋰干預(yù)(lps-10mgli組、lps-20mgli組)海馬局部場電位ripple震蕩功率譜顯著降低,se潛伏期明顯延長,lps-40mgli組大鼠se潛伏期有所延長但無統(tǒng)計學(xué)意義(p0.05),而lps-80mgli組ripple震蕩功率譜密度及振幅明顯增高,se潛伏期明顯縮短(p0.05)。同時與LPS組比較,LPS-10mgLi、LPS-20mgLi組、LPS-VPA組大鼠海馬組織中p-GSK-3β/GSK-3β比值明顯變大(p0.05),海馬組織中致炎因子IL-1β、TNF-α的含量顯著降低(p0.05),抗炎因子IL-10顯著升高(p0.05),伴隨較少激活的小膠質(zhì)細胞(p0.05),海馬CA1區(qū)神經(jīng)元出現(xiàn)排列致密、整齊、脫失少、形狀清楚,染色較深,神經(jīng)元形狀更完整;而LPS-80mg Li組、LPS-WT組出現(xiàn)相反結(jié)果。結(jié)論:LPS可誘導(dǎo)SD大鼠腦炎改變,并導(dǎo)致大鼠CA1區(qū)神經(jīng)元丟失,小膠質(zhì)細胞活化;顱內(nèi)炎癥促進癲癇發(fā)作,小劑量的鋰具有抑制炎性活動,抑制小膠質(zhì)細胞的增生作用,減輕腦炎后腦損傷。大劑量的鋰則作用相反。小劑量鋰鹽可延長腦炎后癲癇放電潛伏期,降低ripple震蕩振幅,降低海馬神經(jīng)元興奮性,提高癲癇發(fā)作閾值,降低腦炎后癲癇發(fā)作的敏感性,對腦炎后癲癇可能具有一定的修飾作用。小劑量鋰鹽抑制大鼠海馬組織中GSK-3β的表達,抑制炎癥因子,減輕組織病理損傷,提示GSK-3β可能參與鋰修飾腦炎后癲癇的機制。
[Abstract]:Objective: to construct an adult SD rat model of encephalitis by injection of lipopolysaccharide (LPS) in the lateral ventricle and detect the changes in the expression of inflammatory factors and GSK-3 beta in the hippocampus. The effects of different doses of lithium salt on the pathophysiological changes of the brain tissue, the expression of GSK-3 beta and the sensitivity of epileptic seizures in the model rats were observed. The indirect inhibition of the GSK-3 beta agonist WT was used. The effect of GSK-3 beta in the epileptic seizures after encephalitis and encephalitis was observed by VPA. The effect and possible mechanism of lithium salt on the seizures of encephalitis and encephalitis were preliminarily discussed. Methods: (1) the preparation of LPS rat model of intracranial infection: 20 adult male SD rats were randomly divided into control Group and group LPS, 10 rats in each group were injected with LPS50 mu g/5ul in the lateral ventricle of group LPS, and the control group was injected with the same volume of physiological saline (NS). After 24 hours of administration, 5 rats were randomly selected to extract the brain, separate the bilateral hippocampal tissues, extract the protein, detect the IL-1 beta and TNF- alpha by ELISA, and detect P-GSK-3 beta and GSK-3 beta by Western-blot method. The remaining 5 rats were perfused to take the brain, and the paraffin sections were used as immunohistochemistry to detect the pathological changes of brain tissue. (2) the effects of different doses of lithium salts on LPS induced inflammation and seizures: 48 adult male SD rats were randomly divided into 6 groups: control group, group LPS, group LPS-10mg Li, group LPS-20mgLi, LPS-40mgLi group, LPS-80mgLi group, and 8 rats in each group, with 8 rats in each group, using 8 rats in each group. The lateral ventricle of the model group of encephalitis was injected with LPS 50 mu g/5 Mu L, and the control group injected the same volume of physiological saline.3 hours after.3 hours, respectively, and injected 5ml/kg in normal saline (NS), Licl of NS, 10,20,40,80mg/Kg, once every 24 hours for 3 days. Then the intraperitoneal injection of pilocarpine (Pilo) was performed. (3) different agents were observed. The effect and possible mechanism of lithium salt on hippocampal pathophysiology and epileptic seizures in LPS rats: 126 adult male SD rats were randomly divided into group lps-10mgli, group lps-20mgli, group lps-40mgli, lps-80mgli group, lps-vpa group, lps-wt group and LPS group, 18 rats in each group, and the lateral ventricle of encephalitis model group was injected lps50 mu g/5 L, 3 The intraperitoneal injection of 10,20,40,80mg/kglicl, 30mg/kgvpa, 0.6 u g/kgwt and 5ml/kg saline, once every 24 hours, for 3 days for 3 days. Each group randomly selected 8 rats by intraperitoneal injection of pilo to observe the basic EEG spectrum of intracranial electroencephalogram, spectrum, changes of high frequency vibration after seizures and the latency and severity of epileptic seizures In addition, each group selected 5 rats randomly to pick up bilateral hippocampal tissues and extract the protein. The concentration of IL-1 beta, tnf- a, IL-10 was detected by ELISA, and the expression of p-gsk-3 beta and GSK-3 beta was detected by Western-blot. The remaining rats were immunohistochemistry to detect the expression of hippocampal neurons and microglia. Results: (1) compared with the control group, the group of LPS group was larger than the control group. The concentration of IL-1 beta and tnf- alpha in the hippocampus increased significantly (P0.05) in the hippocampus (P0.05), and in the LPS group, the se latency was significantly shortened (P0.05), and the content of GSK-3 beta in the hippocampus of the rats was significantly higher than that in the control group, but the content of p-gsk-3 beta was significantly lower than that in the control group (P0.05); C in the hippocampus; C in the hippocampus. There were a large number of activated microglia cells in the A1 area. The neurons were loosely arranged, disordered, lost and blurred, and the morphology was shallow, and the morphology was not complete. Compared with the control group, there were significant differences (P0.05). (2) the local field potential ripple shock power spectrum of the hippocampus decreased significantly in the small dose lithium intervention (group lps-10mgli, lps-20mgli group), and the se latent period was clear. In group lps-40mgli, the latency of Se was prolonged, but there was no statistical significance (P0.05), while the power spectrum density and amplitude of ripple concussion in the lps-80mgli group increased significantly and the latency of Se shortened obviously (P0.05). Meanwhile, the ratio of p-GSK-3 beta beta in the hippocampus of rats of LPS group was significantly higher than that in LPS group. The content of inflammatory factors IL-1 beta, TNF- a in hippocampus decreased significantly (P0.05), anti inflammatory factor IL-10 increased significantly (P0.05), with less activated microglia (P0.05), the neurons of the hippocampal CA1 region appeared dense, neatly, less lost, clear in shape, deep in color, and more complete in the shape of neurons, while LPS-80mg Li group, LPS-WT group appeared opposite. Results. Conclusion: LPS can induce the changes of encephalitis in SD rats, and lead to the loss of neurons in the CA1 area and the activation of microglia, the intracranial inflammation promotes the seizure. The small dose of lithium can inhibit the inflammatory activity, inhibit the proliferation of microglia and reduce the brain injury after encephalitis. The small dose lithium salt can prolong the brain. The latent period of epileptic discharge after inflammation reduces the amplitude of ripple oscillation, reduces the excitability of hippocampal neurons, increases the threshold of epileptic seizures, reduces the sensitivity of epileptic seizures after encephalitis, and may have some modifier effect on postencephalitis epilepsy. Small dose of lithium salt inhibits the expression of GSK-3 beta in the hippocampus of rats, inhibits inflammatory factors and reduces histopathology. Injury suggests that GSK-3 beta may be involved in the mechanism of lithium modifying epilepsy after encephalitis.
【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R742.1

【參考文獻】

相關(guān)期刊論文 前1條

1 Shaoli Wang;Jingyun Zhang;Tao Sheng;Wei Lu;Dengshun Miao;;Hippocampal ischemia causes deficits in local field potential and synaptic plasticity[J];The Journal of Biomedical Research;2015年05期

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本文編號：1924335