發(fā)布時(shí)間：2018-05-08 09:33

  本文選題：苦參素 + 實(shí)驗(yàn)性自身免疫性腦脊髓炎　； 參考：《鄭州大學(xué)》2014年碩士論文

【摘要】：目的： 觀察苦參素（Matrine，MAT）對(duì)多發(fā)性硬化癥（multiple sclerosis，MS）動(dòng)物模型實(shí)驗(yàn)性自身免疫性腦脊髓炎(experimental autoimmune encephalomyelitis，EAE)大鼠臨床癥狀、組織病理學(xué)的影響，分析大鼠發(fā)病過程中血腦屏障體系(blood-brain barrier，BBB)相關(guān)因子如IV型膠原蛋白（Collagen IV），閉鎖小帶蛋白-1（Zonula occludens-1， ZO-1），基質(zhì)金屬蛋白酶-2（matrixmetalloproteinase-2，MMP-2），基質(zhì)金屬蛋白酶-9（matrix metalloproteinase-9，MMP-9），基質(zhì)金屬蛋白酶抑制劑-1（tissue inhibitors of metalloproteinase-1，TIMP-1），基質(zhì)金屬蛋白酶抑制劑-2（tissue inhibitors of metalloproteinase-2，TIMP-2）；谷氨酸興奮系統(tǒng)相關(guān)因子谷氨酸（glutamate，Glu），γ-氨基丁酸（γ-aminobutyric acid，GABA），谷氨酸轉(zhuǎn)運(yùn)體-1（glutamate transport-1，GLT-1），谷氨酸/天冬氨酸轉(zhuǎn)運(yùn)體（glutamate/aspartate transporter，GLAST），N-甲基-D-天冬氨酸受體（N-methyl-d-aspartic acid receptor，NMDAR），α-氨基羥甲基惡唑丙酸受體(α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor，AMPAR)；軸突生長抑制因子-A（Nogo-A）及軸突生長抑制因子受體（Nogoreceptor，NgR）表達(dá)的變化，探究苦參素對(duì)EAE的防治效果及作用機(jī)制，旨在為多發(fā)性硬化癥提供一種安全、有效、經(jīng)濟(jì)的治療藥物。 方法： 1建立Wistar大鼠EAE模型：在無菌條件下制備豚鼠全脊髓勻漿（guinea pigspinal cord homogenate，GPSCH），與等體積含6mg/ml卡介苗的完全弗氏佐劑（complete freund adjuvant，CFA）制備穩(wěn)定油包水型抗原乳劑作為抗原，給予Wistar雌性大鼠足墊皮內(nèi)注射誘導(dǎo)EAE模型。自免疫日起，詳細(xì)觀察并記錄大鼠體重變化、飲食情況、活動(dòng)能力變化情況、臨床癥狀及神經(jīng)功能評(píng)分。 2動(dòng)物分組及給藥：將40只6～8周齡免疫后的Wistar雌性大鼠采用隨機(jī)數(shù)字表法分為四組：EAE模型組（Vehicle）、MAT低劑量組（MAT-L）、MAT高劑量組（MAT-H）、地塞米松組（DEX），每組10只。同時(shí)有10只健康大鼠作為健康對(duì)照組(Naive)。從免疫當(dāng)日起，五組大鼠分別給予不同干預(yù)措施，其中MAT低劑量組每日給予1次6.7ml/kg(150mg/kg)苦參素注射液腹腔注射治療，MAT高劑量組每日給予1次6.7ml/kg(200mg/kg)苦參素注射液腹腔注射治療，DEX組每日給予1次6.7ml/kg(1mg/kg)地塞米松注射液腹腔注射治療，正常對(duì)照組和模型組采用等量生理鹽水替代治療，，五組大鼠均連續(xù)干預(yù)16天。 3動(dòng)物標(biāo)本收集：在免疫第17天，使用10%水合氯醛麻醉后，眼眶后靜脈叢取血3ml，制血清備用；預(yù)冷生理鹽水心臟灌流完全后，留取大鼠腦與脊髓標(biāo)本，部分用于制作石蠟切片，其余立即投入液氮留存?zhèn)溆谩?4相關(guān)指標(biāo)檢測：蘇木素-伊紅（HE）和變色酸-2R-亮綠（C-2R-BG）染色觀察炎性細(xì)胞浸潤及脫髓鞘情況，免疫組織化學(xué)法分析MMP-2、TIMP-2、NMDAR、AMPAR、Nogo-A表達(dá)，酶聯(lián)免疫吸附法（ELISA法）檢測MMP-9、TIMP-1、Glu、GABA水平，逆轉(zhuǎn)錄聚合酶鏈反應(yīng)法（RT-PCR法）分析CollagenIV、ZO-1、GLT-1、GLAST、NgR含量。 結(jié)果： 1發(fā)病率：每組10只動(dòng)物中，正常對(duì)照組大鼠未觀察到發(fā)病情況，模型組有8只發(fā)病，MAT-L組有6只發(fā)病，MAT-H組有5只發(fā)病，DEX組有4只發(fā)病。Fisher確切概率分析結(jié)果示：正常對(duì)照組發(fā)病率顯著低于模型組(p 0.05)；MAT-L組、MAT-H組、DEX組大鼠發(fā)病率均低于模型組，但是差異不具有統(tǒng)計(jì)學(xué)意義(p0.05)。此外三個(gè)藥物干預(yù)組間亦不存在顯著性差異(p0.05)。 2體重變化情況：正常對(duì)照組大鼠自免疫第2天起平均體重穩(wěn)步上升，其余四組平均體重總體上均呈現(xiàn)上升后下降趨勢。兩個(gè)MAT干預(yù)組體重降低幅度顯著低于模型組(p 0.01)，亦低于地塞米松組(p 0.01)。且與MAT-L組相比，MAT-H組大鼠平均體重變化較小，差異具有統(tǒng)計(jì)學(xué)意義(p 0.05)。 3神經(jīng)功能學(xué)評(píng)分：本研究中正常對(duì)照組平均神經(jīng)評(píng)分一直保持為0。免疫后第17天，模型組大鼠平均神經(jīng)功能學(xué)評(píng)分顯著高于三個(gè)藥物干預(yù)組(p 0.05)。三個(gè)藥物干預(yù)組間平均神經(jīng)功能評(píng)分無顯著性差異(p0.05)。 4組織病理學(xué)情況：模型組平均炎癥浸潤評(píng)分和髓鞘脫失評(píng)分較正常對(duì)照組明顯升高，差異具有顯著性意義(p 0.01)。MAT-L組平均炎癥浸潤評(píng)分和髓鞘脫失評(píng)分顯著低于模型組(p 0.01)。與MAT-L組相比，MAT-H組平均炎癥浸潤評(píng)分和髓鞘脫失評(píng)分進(jìn)一步降低(p 0.05)。與MAT-H組相比，DEX組平均炎癥浸潤評(píng)分顯著性降低(p 0.01)，髓鞘脫失評(píng)分無顯著性差異(p0.05)。 5脊髓中Collagen IV、ZO-1mRNA的表達(dá)：與正常對(duì)照組相比，模型組Collagen IV，ZO-1mRNA含量顯著性降低(p 0.01)。兩個(gè)MAT干預(yù)組CollagenIV，ZO-1mRNA表達(dá)均較模型組明顯升高(p 0.01)，其中MAT-H組優(yōu)于MAT-L組(p 0.01)。MAT-H組、DEX組在Collagen IV，ZO-1mRNA表達(dá)方面均無顯著性差異(p0.05)。 6血清中MMP-9、TIMP-1水平：與正常對(duì)照組相比，模型組大鼠血清中MMP-9含量明顯升高(p 0.01)。兩個(gè)MAT干預(yù)組MMP-9水平均較模型組顯著性降低(p 0.01)，其中MAT-H組降低幅度更為明顯(p 0.01)。DEX組在抑制MMP-9病理性升高方面優(yōu)于MAT-H組，差異存在統(tǒng)計(jì)學(xué)意義(p 0.01)。模型組較正常對(duì)照組血清中TIMP-1含量明顯降低(p 0.01)。DEX組TIMP-1水平均較模型組顯著性增高(p 0.01)；MAT升高EAE大鼠血清TIMP-1的能力存在劑量依賴性(p 0.01)。與MAT-H組相比，DEX組TIMP-1水平較低，差異具有統(tǒng)計(jì)學(xué)意義(p 0.01)。 7脊髓中MMP-2、TIMP-2含量：模型組較正常對(duì)照組脊髓中MMP-2含量明顯升高(p 0.01)。兩個(gè)MAT干預(yù)組MMP-2水平均較模型組顯著性降低(p 0.01)，其中MAT-H組降低幅度更為明顯(p 0.05)。DEX組MMP-2表達(dá)低于MAT-H組，但差異不具有統(tǒng)計(jì)學(xué)意義(p0.05)。與正常對(duì)照組相比，模型組大鼠血清中TIMP-2含量明顯減低(p 0.01)。三個(gè)藥物組TIMP-2水平均較模型組顯著性增高(p 0.05)。MAT升高EAE大鼠血清TIMP-1的能力存在劑量依賴性(p 0.01)。MAT-H組與DEX組在TIMP-2含量方面無顯著性差異(p0.05)。 8大腦皮質(zhì)Glu、GABA含量：與正常對(duì)照組相比，模型組大鼠腦皮質(zhì)Glu含量明顯升高(p 0.01)。MAT-L組Glu含量較模型組低，但是差異不具有統(tǒng)計(jì)學(xué)意義(p0.05)。MAT-H組與DEX組Glu含量均比模型組低(p 0.05)，但是兩者間無顯著性差別(p0.05)。模型組大鼠腦皮質(zhì)GABA含量比正常對(duì)照組顯著性降低(p 0.01)。MAT升高EAE腦皮質(zhì)GABA的能力存在劑量依賴性(p 0.05)。與DEX組相比，MAT-L組GABA水平較低，差異具有統(tǒng)計(jì)學(xué)意義(p 0.05)。MAT-H組與DEX組在腦皮質(zhì)GABA含量方面不存在顯著性差異(p0.05)。 9大腦皮質(zhì)GLT-1、GLAST mRNA表達(dá)：與正常對(duì)照組相比，模型組GLT-1，GLAST mRNA含量顯著降低(p 0.01)；三個(gè)藥物干預(yù)組GLT-1mRNA表達(dá)均較模型組顯著性升高(p 0.01)，但組間差異不具有顯著性(p0.05)。MAT-L組和MAT-H組GLAST mRNA表達(dá)均較模型組明顯升高(p 0.01)，其中MAT-H組更高，但差異不具有統(tǒng)計(jì)學(xué)意義(p0.05)。高劑量MAT在提高GLAST mRNA含量方面優(yōu)于DEX (p 0.05)。 10大腦皮質(zhì)NMDAR、AMPAR表達(dá)：模型組較正常對(duì)照組大腦皮質(zhì)中NMDAR、AMPAR含量明顯升高(p 0.01)。兩個(gè)MAT干預(yù)組NMDAR、AMPAR水平均較模型組顯著性降低(p 0.05)，其中MAT-H組降低幅度更為明顯(p 0.01)。MAT-H組AMPAR表達(dá)低于DEX組，差異具有統(tǒng)計(jì)學(xué)意義(p 0.05)；但是兩組在NMDAR表達(dá)方面無統(tǒng)計(jì)學(xué)差異(p0.05)。 11脊髓中Nogo-A蛋白、NgR mRNA表達(dá)：模型組較正常對(duì)照組脊髓中Nogo-A蛋白，NgR mRNA含量明顯升高(p 0.01)。兩個(gè)MAT干預(yù)組Nogo-A蛋白，NgR mRNA水平均較模型組顯著性降低(p 0.01)，其中MAT-H組降低幅度更為明顯(p 0.01)。MAT-H組Nogo-A蛋白，NgR mRNA表達(dá)高于DEX組，差異具有統(tǒng)計(jì)學(xué)意義(p 0.01)。 結(jié)論： MAT對(duì)Wistar大鼠EAE具有良好的防治作用，可能是通過上調(diào)機(jī)體血腦屏障基質(zhì)膜、緊密連接蛋白，維持體內(nèi)MMPs與TIMPs的平衡；下調(diào)谷氨酸及其受體含量，提高其天然抑制劑GABA和谷氨酸轉(zhuǎn)運(yùn)體的表達(dá)；抑制Nogo-A及受體NgR水平，發(fā)揮保護(hù)大鼠血腦屏障、抑制谷氨酸系統(tǒng)過度激活、下調(diào)Nogo-A/NgR軸索抑制通路興奮性作用，從而起到對(duì)EAE大鼠的防治效果。
[Abstract]:Purpose :

To observe the effects of Matrine ( MAT ) on the clinical symptoms and histopathological changes in rats with multiple sclerosis ( MS ) models and to analyze the effects of matrix metalloproteinase - 2 ( MMP - 2 ) , matrix metalloproteinase - 9 ( MMP - 9 ) , matrix metalloproteinase - 1 ( MMP - 2 ) , matrix metalloproteinase - 9 ( MMP - 9 ) , matrix metalloproteinase - 1 ( MMP - 2 ) , matrix metalloproteinase - 9 ( TIMP - 1 ) , matrix metalloproteinase - 2 ( tissue inhibitors of metalloproteinase - 2 , TIMP - 2 ) .
glutamate ( glutamate , Glu ) , gamma - aminobutyric acid ( GABA ) , glutamate transporter - 1 ( glutamate transport - 1 , GLT - 1 ) , glutamate / aspartate transporter ( GLAST ) , N - methyl - D - aspartate receptor ( N - methyl - d - glutamic acid receptor , NMDAR ) , 偽 - amino - methyloxacillin - propionic acid receptor ( AMPAR ) ;
Objective To explore the effect and mechanism of matrine on the prevention and treatment of multiple sclerosis and to provide a safe , effective and economical drug for multiple sclerosis .

Method :

1 Wistar rat model was established : guinea pig whole spinal cord homogenate ( GPSCH ) was prepared under aseptic conditions . Complete freund adjuvant ( CFA ) was prepared by complete freund adjuvant ( CFA ) .

Two groups of rats were randomly divided into four groups : Vehicle , MAT - L , MAT - H and DEX . At the same time , 10 healthy rats were injected intraperitoneally with 6 . 7ml / kg ( 150 mg / kg ) of Matrine injection .

3 Animal specimens collected : After 17 days of immunization , after anesthesia with 10 % chloral hydrate , 3 ml of blood were taken from the posterior vein plexus of the orbit , and the serum was prepared for standby ;
After the pre - cold normal saline heart perfusion was complete , the rat brain and spinal cord specimens were removed , partially used to make paraffin sections , and the rest was put into liquid nitrogen for standby immediately .

The expression of MMP - 2 , TIMP - 2 , NMDAR , AMPAR , Nogo - A and enzyme - linked immunosorbent assay ( ELISA ) were used to detect MMP - 9 , TIMP - 1 , Glu , GABA levels and reverse transcription polymerase chain reaction ( RT - PCR ) .

Results :

1 Incidence rate : In 10 animals in each group , the incidence was not observed in the normal control group , 8 in the model group , 6 in the MAT - L group , 5 in the MAT - H group , 4 in the DEX group , and the Fisher exact probability analysis showed that the incidence of the normal control group was significantly lower than that of the model group ( p 0.05 ) ;
The morbidity of MAT - L group , MAT - H group and DEX group was lower than that in model group , but the difference was not statistically significant ( p < 0.05 ) . There was no significant difference among the three drug intervention groups ( p < 0.05 ) .

2 Weight changes : The average body weight increased steadily in the normal control group from the second day , and the average body weight in the remaining four groups showed a decrease trend . The body weight loss of the two MAT groups was significantly lower than that in the model group ( p 0.01 ) , which was also lower than that of the dexamethasone group ( p 0.01 ) . Compared with MAT - L group , the mean body weight change of MAT - H group was small , and the difference was statistically significant ( p 0.05 ) .

3 Neurologic score : The average nerve score of the normal control group was maintained at 0 . The mean neurological score in the model group was significantly higher than that of the three drug intervention groups ( p 0.05 ) on the 17th day after immunization . There was no significant difference between the three groups ( p < 0.05 ) .

4 histopathological findings : The mean inflammatory infiltration score and myelination score in the model group were significantly higher than those in the normal control group ( p 0.01 ) . The mean inflammatory infiltration score and myelination score of MAT - L group were significantly lower than that of model group ( p 0.01 ) . The mean inflammatory infiltration score and myelination score of MAT - H group were further reduced compared to MAT - L group ( p 0.05 ) . Compared with MAT - H group , the mean inflammatory infiltration score of DEX group was significantly lower ( p 0.01 ) , and there was no significant difference in myelination ( p < 0.05 ) .

5鑴婇珦涓瑿ollagen IV,ZO-1mRNA鐨勮〃杈撅細(xì)涓庢甯稿鐓х粍鐩告瘮,妯″瀷緇凜ollagen IV,ZO-1mRNA鍚噺鏄捐憲鎬ч檷浣

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