發(fā)布時間：2018-04-27 00:27

  本文選題：實驗性自身免疫性腦脊髓炎 + 淫羊藿苷�。� 參考：《廣東藥科大學》2016年碩士論文

【摘要】：【目的】中樞神經(jīng)系統(tǒng)白質脫髓鞘病變是多發(fā)性硬化(multiple sclerosis,MS)的主要病理特點,近年有關MS的星形膠質細胞異常及軸突損害才逐步受到重視。我們的前期研究證實淫羊藿苷(ICA)可通過影響炎性細胞因子、HPA軸途徑調節(jié)炎性免疫機制而發(fā)揮對實驗性自身免疫性腦脊髓炎(EAE)小鼠癥狀緩解及髓鞘保護作用。本研究擬在前期研究的基礎上,繼續(xù)采用以復發(fā)緩解型為特征的雌性C57BL/6 EAE小鼠模型為研究對象,通過觀察治療前后各組間小鼠體重、神經(jīng)功能評分變化以及腦胼胝體切片GFAP(單標)、NF200+β-APP(雙標)免疫熒光染色變化,探討EAE小鼠腦星形膠質細胞異常和軸突損害,并研究ICA對其影響,揭示ICA對EAE是否具有非炎性神經(jīng)保護作用�！痉椒ā康谝徊糠诌x取SPF級65只7-9周齡C57BL/6雌性小鼠,15只作為正常對照組,50只用于造模。將小鼠麻醉后將油包水狀態(tài)的MOG35-55抗原乳劑按每只小鼠300ug的劑量皮下注射接種于小鼠脊柱兩側皮下(分四個部位注射),在接種后當天及48小時后腹腔注射500ng百日咳毒素,初次免疫后第7天再次加強免疫1次。免疫后動態(tài)觀察小鼠是否出現(xiàn)神經(jīng)功能癥狀并對其神經(jīng)功能損害進行評分。在小鼠疾病高峰期隨機抽取3只EAE小鼠及3只正常小鼠處死,取脊髓腰膨大用于HE染色,光鏡下觀察脊髓組織學變化,確定造模是否成功。第二部分將造模成功后的EAE小鼠按評分相當?shù)脑瓌t隨機分配到3組內,分別為雌激素組(陽性對照組)、ICA組、模型對照組,加上正常對照組(空白對照組),共4組,分別給予己烯雌酚0.2mg/kg.d、ICA300mg/kg.d、等容量0.5%羧甲基纖維素鈉、等容量0.5%羧甲基纖維素鈉灌胃,連續(xù)給藥5天,給藥后第6天取材,以多聚甲醛心臟灌注取腦組織,經(jīng)后固定,蔗糖脫水后進行冰凍切片分別進行熒光染色:(1)GFAP(星形膠質細胞標志)熒光單標染色:根據(jù)免疫熒光染色步驟分別加入一抗GFAP,帶紅色熒光的二抗Cy3并封片,在熒光顯微鏡下,GFAP發(fā)紅色熒光;(2)NF200(軸突標志)+β-APP(軸突損害標志)熒光雙標染色:根據(jù)免疫熒光染色步驟加入一抗NF200、β-AP P,分別加入帶綠色熒光的二抗Alexa Fluor488、帶紅色熒光的二抗Cy3并封片,在熒光顯微鏡下,NF200發(fā)綠色熒光,β-APP發(fā)紅色熒光。將以上染色后的切片在40×物鏡熒光顯微鏡下觀察并拍攝照片。采用Image J軟件對GFAP、NF200+β-APP圖片進行熒光強度分析,采用SPSS Statistics 17軟件行統(tǒng)計學分析,應用Graph Pad Prism軟件進行作圖。計量資料采用`x±s表示。多組間比較采用單因素方差分析,P0.05為差異有統(tǒng)計學意義,P0.01為差異有顯著統(tǒng)計學意義。相關趨勢變量采用Pearson相關分析�！窘Y果】第一部分小鼠于免疫后第13-15天開始發(fā)病,2-3天后達疾病高峰,以尾部無力為首發(fā)癥狀的小鼠占大部分,亦有少部分小鼠以行走時平衡障礙發(fā)病,逐漸加重至肢體無力、肢體癱瘓。最高神經(jīng)功能評分達5分,正常對照組小鼠無明顯異常。對EAE小鼠脊髓切片HE染色可見大量淋巴細胞浸潤,以血管周圍顯著,呈“袖套樣”改變。而正常對照組未見病變。根據(jù)小鼠癥狀、神經(jīng)功能評分及HE染色組織學改變說明構建EAE小鼠模型成功。第二部分(1)小鼠體重變化:小鼠接受抗原刺激后,體重開始出現(xiàn)下降,隨著時間的推移,體重下降越來越明顯。給予治療后,雌激素組和ICA組EAE小鼠體重有明顯增加(P0.01),治療后各組與模型對照組比較,雌激素組和ICA組體重有明顯改善(P0.01),雌激素組與ICA組比較無明顯差異(P0.05)。(2)小鼠神經(jīng)功能變化:雌激素組和ICA組EAE小鼠治療后神經(jīng)功能評分有明顯改善(P0.05),治療后各組與模型對照組比較,雌激素組和ICA組神經(jīng)功能評分亦有明顯好轉(P0.05),雌激素組與ICA組比較無明顯差異(P0.05)。(3)EAE小鼠存在星形膠質細胞異常及軸突損害:模型對照組GFAP(星形膠質細胞標志)熒光強度比正常組顯著增高,同時細胞形態(tài)異常,數(shù)量增加,說明EAE小鼠存在星形膠質細胞活化增殖;模型對照組軸突(NF200標記)密度較正常組明顯下降,說明EAE小鼠存在軸突損害。(4)ICA對EAE小鼠星形膠質細胞的影響:正常組的星形膠質細胞細胞體較小,突起如細絲狀向周邊延伸,形態(tài)未見異常,GFAP(星形膠質細胞標志)熒光強度低;模型對照組熒光強度比正常組顯著增高(P0.05),且其處于活化狀態(tài),細胞形態(tài)異常,胞體增大且變得不規(guī)則,突起粗短;與模型對照組相比,雌激素組及ICA組熒光強度明顯降低(P0.05),形態(tài)基本接近正常。雌激素組與ICA組比較無明顯差異(P0.05)。(5)ICA對EAE小鼠軸突的影響:模型對照組軸突(NF200標記)密度較正常組明顯下降(P0.01);雌激素、ICA治療組軸突密度較模型對照組明顯增高,均具有統(tǒng)計學意義,其中ICA組增高更明顯(P0.01),雌激素組與ICA組之間未見明顯差異(P0.05)。而在正常組未見損害軸突(β-APP標記),模型對照組β-APP熒光強度最高,表明軸突損害最嚴重,與模型對照組比較,經(jīng)治療后雌激素組和ICA組β-APP熒光強度均有明顯下降(P0.01),雌激素組與ICA組之間未見明顯差異(P0.05)。【結論】(1)EAE小鼠胼胝體內存在星形膠質細胞異常,表現(xiàn)為增殖活化狀態(tài),細胞形態(tài)異常,數(shù)量增加;且同時存在軸突損害,表現(xiàn)為軸突密度下降;(2)ICA治療后,EAE小鼠胼胝體內星形膠質細胞活化減少,ICA可能通過抑制星形膠質細胞活化增殖來改善模型鼠神經(jīng)功能損害;(3)ICA對EAE小鼠具有非炎性神經(jīng)保護作用,可能與其調節(jié)軸突再生,減少軸突損害密切相關。
[Abstract]:[Objective] the central nervous system white matter demyelination is the main pathological feature of multiple sclerosis (MS). In recent years, the astrocyte abnormalities and axonal damage of MS have been gradually paid attention to. Our previous study confirmed that icariin (ICA) can affect inflammatory cytokines, and HPA axis pathway regulates inflammatory immunity. On the basis of the previous study, this study intends to continue to use the female C57BL/6 EAE mouse model characterized by remission type on the basis of the previous study. By observing the weight of mice and the changes of nerve function score in each group before and after the treatment. And the changes of GFAP (Dan Biao) and NF200+ beta -APP (double standard) immunofluorescence staining of the corpus callosum, explore the abnormalities of astrocyte and axon damage in the brain of EAE mice, and study the effect of ICA on it, and reveal whether ICA has non inflammatory neuroprotective effect on EAE. [Methods] the first part selected 65 7-9 weeks old C57BL/6 female mice at SPF level and 15 mice. For the normal control group, 50 rats were used to build the model. After the mice were anesthetized, the MOG35-55 antigen emulsion was injected subcutaneously on the two sides of the spinal column at the dose of 300ug in each mouse (four parts of the mice). The peritoneal injection of 500ng 100 day cough toxin was intraperitoneally injected after the day of inoculation and 48 hours after the inoculation, and the immunization was strengthened again after the first immunization. The 1 time. After immunization, the neurological function symptoms were observed and the neurological impairment was evaluated. 3 EAE mice and 3 normal mice were killed at the peak period of the disease. The spinal lumbar enlargement was used for HE staining. The histological changes of the spinal cord were observed under the light microscope to determine whether the model was successful. The second part would succeed in making the model. After the EAE mice were randomly assigned to 3 groups according to the principle of score, they were estrogen group (positive control group), group ICA, model control group, plus normal control group (blank control group), total of 4 groups, respectively given diethylstilbestrol 0.2mg/kg.d, ICA300mg/kg.d, 0.5% carboxymethyl cellulose sodium, equal capacity of 0.5% carboxymethyl cellulose sodium gavage. After 5 days of administration, the brain tissue was taken for sixth days after administration, and the brain tissue was perfused with paraformaldehyde at the heart. After being immobilized, the frozen sections were stained respectively after the sucrose was dehydrated: (1) GFAP (astrocyte marker) fluorescence single mark staining: according to the immunofluorescence staining steps, a anti GFAP, two anti Cy3 with red fluorescence and the fluorescence were added to the fluorescence. Under the microscope, GFAP hair red fluorescence; (2) NF200 (axon sign) + beta -APP (axon damage marker) fluorescent double labeling staining: according to immunofluorescence staining steps added to a anti NF200, beta -AP P, adding green fluorescence of the two anti Alexa Fluor488, red fluorescent two anti Cy3 and seal, under the fluorescence microscope, NF200 hair green fluorescence, beta -APP hair Red fluorescence. The above stained slices were observed and photographed under the 40 * objective microscope fluorescence microscope. Image J software was used to analyze the fluorescence intensity of GFAP and NF200+ beta -APP images. The SPSS Statistics 17 software was used for statistical analysis and Graph Pad Prism software was used for mapping. Using single factor analysis of variance, P0.05 was statistically significant and P0.01 had significant statistical significance. The correlation trend variable adopted Pearson correlation analysis. [results] the first part of the first part of the mice began to attack on the 13-15 day after the immunization, and reached the peak of the disease in 2-3 days. The mice were divided by walking balance disorder, and gradually increased to limb weakness and limb paralysis. The maximum nerve function score was 5 points. There was no obvious abnormality in the normal control group. A large number of lymphocyte infiltration was observed in the spinal section of EAE mice with HE staining, and the "sleeve like" changes were observed around the blood vessels, but the normal control group had no pathological changes. The mice symptoms, the neurological function score and the HE staining histological changes showed that the model of the EAE mice was successful. Second (1) the body weight changes in mice: the weight of mice began to decrease after receiving the antigen stimulation, and the weight loss became more and more obvious as time went on. After treatment, the weight of EAE mice in the estrogen group and the ICA group increased significantly (P0. 01), compared with the model control group, the weight of estrogen group and ICA group was significantly improved (P0.01), and there was no significant difference between the estrogen group and the ICA group (P0.05). (2) the changes of neural function in the mice were significantly improved (P0.05) after the treatment of the estrogen group and the ICA group of EAE mice (P0.05). The female group was compared with the model control group after treatment. There was a significant improvement in nerve function score in both group and ICA group (P0.05). There was no significant difference between the estrogen group and the ICA group (P0.05). (3) there was astrocyte abnormality and axon damage in EAE mice: the fluorescence intensity of GFAP (astrocyte marker) in the model control group was significantly higher than that in the normal group, and the cell morphology was abnormal and the number was increased, indicating that the EAE was small. The rat astrocytes were activated and proliferated; the density of the axon (NF200 markers) in the model control group was significantly lower than that in the normal group, indicating the axon damage in the EAE mice. (4) the effect of ICA on astrocytes in EAE mice: the astrocytes in the normal group were smaller, and the protuberances extended like the filaments to the periphery, and the morphology was not abnormal, GFAP (Star gum) The fluorescence intensity of the stromal cells was low, and the fluorescence intensity of the model control group was significantly higher than that of the normal group (P0.05), and it was in the activation state, the cell morphology was abnormal, the cell body increased and became irregular and the protuberance was short. Compared with the model control group, the fluorescence intensity of the estrogen group and the ICA group decreased significantly (P0.05), and the morphology was almost normal. Estrogen group and IC were almost normal. There was no significant difference in the A group (P0.05). (5) the effect of ICA on the axon of EAE mice: the density of the axon (NF200 marker) in the model control group was significantly lower than that in the normal group (P0.01). The axon density in the estrogen and ICA treatment group was significantly higher than that in the model control group, and all of them were statistically significant, and the increase of ICA group was more obvious (P0.01), and no between the estrogen group and the ICA group. There was no significant difference (P0.05) in the normal group (beta -APP). The fluorescence intensity of beta -APP in the model control group was the highest, indicating that the axon damage was the most serious. Compared with the model control group, the fluorescence intensity of the beta -APP in the estrogen group and the ICA group decreased significantly after treatment (P0.01), and there was no significant difference between the estrogen group and the ICA group (P0.05). (1) there was an abnormal astrocyte in the corpus callosum of EAE mice, which showed proliferation activation state, abnormal cell morphology, increasing number, and axon damage, showing a decrease in axon density. (2) after ICA treatment, the activation of astrocytes in the corpus callosum of EAE mice decreased, and ICA could inhibit the proliferation of astrocytes by inhibiting astrocytes. (3) ICA has a non inflammatory neuroprotective effect on EAE mice, which may be related to the regulation of axonal regeneration and axonal damage.

【學位授予單位】：廣東藥科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R277.7

【相似文獻】

相關期刊論文 前10條

1 項紅兵,田玉科;星形膠質細胞與疼痛敏化調控[J];臨床麻醉學雜志;2003年09期

2 劉慧,王小軍,胡榮,楊忠,蔡文琴;星形膠質細胞[J];生理科學進展;2004年01期

3 張敬軍;;星形膠質細胞的研究[J];中國藥理學通報;2006年07期

4 陳彬;劉寬;王偉;;成年大鼠神經(jīng)元和星形膠質細胞細胞周期蛋白依賴性激酶抑制因子表達的差異研究[J];卒中與神經(jīng)疾病;2007年05期

5 沈維高;何欣;王振江;;星形膠質細胞的生物學功能及其與疾病的關系研究進展[J];北華大學學報(自然科學版);2008年06期

6 ;英國科學家確定星形膠質細胞新功能[J];中國當代醫(yī)藥;2010年27期

7 靳哲;趙忠新;;星形膠質細胞在癲癇發(fā)病中的作用研究[J];世界臨床藥物;2012年01期

8 李軍杰,張致身;短暫性腦缺血發(fā)作后海馬區(qū)星形膠質細胞形態(tài)學研究[J];首都醫(yī)科大學學報;1998年04期

9 解旭東,田國紅;星形膠質細胞與癲癇[J];中風與神經(jīng)疾病雜志;1999年01期

10 莫永炎,姜勇,陳瑗;腦星形膠質細胞生物學功能研究進展[J];生理科學進展;2002年01期

相關會議論文 前10條

1 劉慶瑩;趙珠峰;朱長庚;;星形膠質細胞在癲癇復發(fā)中的作用[A];解剖學雜志——中國解剖學會2002年年會文摘匯編[C];2002年

2 唐愛輝;王同飛;王世強;;星形膠質細胞鈣活動:腦細胞網(wǎng)絡中的第二興奮系統(tǒng)?[A];中國神經(jīng)科學學會第六屆學術會議暨學會成立十周年慶祝大會論文摘要匯編[C];2005年

3 池美珠;陳超;錢燕;李劍敏;吳步猛;;產(chǎn)前重復激素治療對星形膠質細胞發(fā)育的影響[A];浙江省圍產(chǎn)醫(yī)學學術會議論文匯編[C];2005年

4 魏爾清;;半胱氨酰白三烯受體對星形膠質細胞功能的調節(jié)作用[A];中國藥理學會第九次全國會員代表大會暨全國藥理學術會議論文集[C];2007年

5 吳建云;王鮮忠;王劍;諶劍波;范光麗;張家驊;;大腦皮質星形膠質細胞培養(yǎng)及鑒定[A];中國畜牧獸醫(yī)學會動物解剖學及組織胚胎學分會第十五次學術研討會論文集[C];2008年

6 趙玉武;程曉娟;;星形膠質細胞與腦水腫的形成[A];山東省2013年神經(jīng)內科學學術會議暨中國神經(jīng)免疫大會2013論文匯編[C];2013年

7 董其平;柴真;;谷氨酸對培養(yǎng)的星形膠質細胞的鈣活動的影響[A];“基因、進化與生理功能多樣性”海內外學術研討會暨中國生理學會第七屆比較生理學學術會議論文摘要[C];2009年

8 曾俊;李康生;;柯薩奇病毒感染星形膠質細胞及細胞因子變化研究[A];新發(fā)和再發(fā)傳染病防治熱點研討會論文集[C];2010年

9 鮑歡;徐曉云;顧曉波;徐霞紅;胡暉;;缺氧后水通道蛋白4在體外培養(yǎng)星形膠質細胞表達變化的實驗研究[A];中華醫(yī)學會第十三次全國神經(jīng)病學學術會議論文匯編[C];2010年

10 熊加祥;白云;宋敏;王艷艷;楊曉亞;;星形膠質細胞表達免疫分子維持大腦免疫穩(wěn)態(tài)[A];中國神經(jīng)科學學會第六屆學術會議暨學會成立十周年慶祝大會論文摘要匯編[C];2005年

相關重要報紙文章 前7條

1 記者 胡德榮;星形膠質細胞由“天使”變“魔鬼”原因找到[N];健康報;2012年

2 記者 劉海英;星形膠質細胞可調節(jié)呼吸強度[N];科技日報;2010年

3 劉霞;腦內星形膠質細胞在實驗室培育成功[N];科技日報;2011年

4 汪敏華;大腦星形膠質細胞有兩種新功能[N];解放日報;2003年

5 吳一福;黃芪可調節(jié)星形膠質細胞“時間模式”[N];中國醫(yī)藥報;2005年

6 南方日報記者 曹斯　實習生 趙莞 張寧 通訊員 鄒瑩 張淼;用ATP對抗不快樂[N];南方日報;2013年

7 記者 班瑋;德國腦細胞再生研究取得新進展[N];人民日報;2010年

相關博士學位論文 前10條

1 曾招;離子通道TRPM7調節(jié)細胞生理的分子機制研究[D];蘇州大學;2014年

2 黃維一;骨髓間充質干細胞旁分泌因子對缺血性腦卒中后星形膠質細胞活性及功能的影響[D];南方醫(yī)科大學;2015年

3 楊俊華;星形膠質細胞來源的ATP促進丘腦中的突觸刪除[D];浙江大學;2015年

4 鄧雅婷;龍膽苦苷鎮(zhèn)痛與抗抑郁的中樞作用及機制研究[D];第四軍醫(yī)大學;2015年

5 李新;星形膠質細胞NDRG2在七氟烷預處理神經(jīng)保護作用中的機制研究[D];第四軍醫(yī)大學;2015年

6 鐘志宏;亞鐵離子對星形膠質細胞的損傷作用及香芹酚在實驗性腦出血中的神經(jīng)保護作用[D];上海交通大學;2014年

7 張建成;星形膠質細胞來源的IL-17A對缺血性腦卒中神經(jīng)再生和功能恢復的影響及機制研究[D];華中科技大學;2015年

8 洪洋;孕酮對Aβ所致星形膠質細胞炎癥反應的調節(jié)作用及機制研究[D];河北醫(yī)科大學;2016年

9 凌云志;帕瑞昔布減輕過氧化氫誘導的大鼠星形膠質細胞氧化應激損傷的作用及機制研究[D];安徽醫(yī)科大學;2015年

10 徐進;腦出血后星形膠質細胞AQP4內化及其機制的研究[D];重慶醫(yī)科大學;2015年

相關碩士學位論文 前10條

1 龔佩佩;磷酸化MSK1在星形膠質細胞炎癥活化過程中的作用的研究[D];南通大學;2013年

2 何潔玉;糖皮質激素對星形膠質細胞分泌GnRH的調控機制[D];西南大學;2015年

3 張麗麗;脂肪基質細胞誘導分化為星形膠質細胞過程中線粒體凋亡與自噬的關系[D];河北聯(lián)合大學;2014年

4 燕茹;高同型半胱氨酸血癥致ApoE~(-/-)小鼠腦血管、星形膠質細胞及神經(jīng)元損傷作用研究[D];寧夏醫(yī)科大學;2015年

5 米鵬霞;P物質對星形膠質細胞的活化作用及機制研究[D];山西醫(yī)科大學;2015年

6 楊志奇;小鼠皮層內移植膠質細胞形態(tài)及功能重建研究[D];第三軍醫(yī)大學;2015年

7 陳雅南;敲除星形膠質細胞的dicer對中樞神經(jīng)系統(tǒng)的影響[D];杭州師范大學;2015年

8 馬輝;星形膠質細胞上酸敏感離子通道1a在顳葉癲癇發(fā)生中的作用研究[D];第四軍醫(yī)大學;2015年

9 周亞蘭;IFN-γ介導T細胞參與炎性痛慢性化機制的研究[D];第二軍醫(yī)大學;2015年

10 鄧子輝;瘦素抑制星形膠質細胞縫隙連接蛋白-43表達改善腦缺血再灌注損傷的機制研究[D];中國人民解放軍醫(yī)學院;2015年

，

本文編號：1808378