本文選題：多發(fā)性硬化　切入點(diǎn)：馬索羅酚和丹參酮ⅡA　出處：《河北醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：中樞神經(jīng)系統(tǒng)脫髓鞘疾病是部位發(fā)生在腦和脊髓，主要特征是髓鞘損傷，而神經(jīng)元胞體和軸索損害相對(duì)較輕的一組疾病。多發(fā)性硬化（Multiple sclerosis, MS）是由免疫介導(dǎo)的中樞神經(jīng)系統(tǒng)慢性炎性脫髓鞘性疾病，病因及發(fā)病機(jī)制迄今不明，最新研究表明，MS的病因與遺傳因素和環(huán)境因素有關(guān),而疾病導(dǎo)致的髓鞘脫失和軸索變性是患者表現(xiàn)為神經(jīng)功能缺損，病程呈現(xiàn)為緩解-復(fù)發(fā)和病情持續(xù)進(jìn)展的主要原因。實(shí)驗(yàn)性自身免疫性腦脊髓炎(Experimental autoimmune encephalomyelitis, EAE)是MS經(jīng)典的動(dòng)物模型，其病程上可以表現(xiàn)為復(fù)發(fā)-緩解，很接近于人類MS的臨床表現(xiàn)及病理特征。本研究應(yīng)用MOG35-55建立EAE動(dòng)物模型，在EAE小鼠出現(xiàn)神經(jīng)癥狀后開始給予馬索羅酚和丹參酮IIA治療，模擬臨床的治療時(shí)間，通過HE染色觀察兩種藥物對(duì)EAE動(dòng)物模型是否有抗炎作用，通過羅克沙爾堅(jiān)牢藍(lán)（Luxol Fast Blue）特殊髓鞘染色和Bielschowsky's神經(jīng)軸索染色從病理學(xué)和形態(tài)學(xué)的角度觀察馬索羅酚和丹參酮IIA是否能夠減輕CNS髓鞘的脫失，減輕軸索病理?yè)p傷，降低EAE小鼠的神經(jīng)功能評(píng)分。 方法：本研究采用了45只8-12周齡，健康C57BL/6雌性小鼠，小鼠隨機(jī)分成三組即EAE組、NDGA組和DANIIA組，以MOG35-55為抗原免疫C57BL/6小鼠建立EAE模型。用生理鹽水將抗原MOG35-55稀釋成為5mg/ml的液體狀態(tài)，然后將完全弗氏佐劑（其中包含的結(jié)核桿菌H37Ra的終濃度為4mg/ml）按1:1的體積加入并混合均勻，乳化后按每只0.1ml在小鼠脊柱兩側(cè)的皮膚下分四點(diǎn)注射，免疫后的當(dāng)天即第0h及第48h腹腔內(nèi)注射500ngPTX（1000ng/只）。小鼠發(fā)病后，自神經(jīng)功能評(píng)分達(dá)到1分以上時(shí)開始給予藥物治療，即EAE組小鼠腹腔注射5%DMSO，NDGA組小鼠腹腔注射馬索羅酚，DANIIA組小鼠腹腔注射丹參酮，分別于治療前，治療10d，治療20d取材，每組各取5只。取材部位為小鼠脊髓腰膨大處，組織進(jìn)行石蠟包埋，分別通過HE染色觀察病變部位炎性反應(yīng)程度和血管套形成的情況，羅克沙爾堅(jiān)牢藍(lán)（Luxol Fast Blue）特殊髓鞘染色和Bielschowsky's神經(jīng)軸索染色從病理學(xué)及形態(tài)學(xué)的角度觀察馬索羅酚和丹參酮IIA兩種藥物能否可以減輕小鼠病變部位的髓鞘脫失，減輕組織的軸索損傷，降低EAE小鼠的神經(jīng)功能評(píng)分起到治療作用。應(yīng)用SPSS19.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)學(xué)處理，結(jié)果為計(jì)量資料則均數(shù)±標(biāo)準(zhǔn)差(±S)表示，而多組計(jì)量資料均數(shù)的比較則采用ANOVA方差分析，若不滿足正態(tài)分布或方差齊性，則采用秩和檢驗(yàn)的方法，P0.05表明差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果： 1動(dòng)物臨床癥狀及評(píng)分觀察： 1.1臨床癥狀： 45只小鼠全部在10-16d內(nèi)開始發(fā)病，表明EAE動(dòng)物模型制作成功，可以給予藥物干預(yù)治療，EAE組小鼠免疫后全部發(fā)病，出現(xiàn)皮毛粗糙、精神萎靡不振、行走困難、喪失食欲、體重減輕等表現(xiàn)。臨床表現(xiàn)最嚴(yán)重在發(fā)病高峰期，小鼠出現(xiàn)雙后肢甚至全部四肢肌力降低甚至全部癱瘓、大便、小便均失禁、不能夠自主翻身，個(gè)別成垂死狀態(tài)，并且持續(xù)較長(zhǎng)時(shí)間。治療組的小鼠免疫后也全部發(fā)病，于癥狀評(píng)分達(dá)到1分以上時(shí)給予藥物干預(yù)，治療前與EAE組臨床癥狀評(píng)分無(wú)顯著差異,治療10天后，此時(shí)EAE處于發(fā)病高峰期，臨床表現(xiàn)嚴(yán)重，而給予丹參酮及馬索羅酚治療的小鼠出現(xiàn)尾部癱瘓或者雙后肢輕癱或全癱，無(wú)大小便失禁及不能自主翻身等癥狀，治療20天后，，EAE小鼠隨著病程發(fā)展，癥狀有所緩解，但仍遺留尾部癱瘓或后肢癱瘓等癥狀，給予丹參酮及馬索羅酚治療的小鼠癥狀緩解較快，臨床癥狀表現(xiàn)更輕，僅表現(xiàn)為尾部輕癱或者尾部無(wú)力等癥狀。 1.2臨床評(píng)分觀察： 計(jì)算并比較各組中每只小鼠于治療10d及治療20d的臨床評(píng)分均數(shù)。EAE組小鼠神經(jīng)功能評(píng)分高于丹參酮組及馬索羅酚組（P0.05）。丹參酮組及馬索羅酚組小鼠神經(jīng)功能評(píng)分相比較，差別沒有統(tǒng)計(jì)學(xué)意義（P＞0.05）。 2HE染色： 2.1治療前三組可見散在炎性細(xì)胞浸潤(rùn)，大部分集中在脊髓白質(zhì)附近。 2.2治療10天EAE組可見大片狀炎性細(xì)胞的浸潤(rùn)，甚至形成了“血管袖套”現(xiàn)象，即脊髓多處小血管周圍，特別是小靜脈四周出現(xiàn)了炎癥細(xì)胞，大部分為淋巴細(xì)胞，病變大多在前角白質(zhì)，灰白質(zhì)交界及前正中裂血管周圍，丹參酮組及馬索羅酚組“血管袖套”形成較少，炎性細(xì)胞主要集中于小血管周圍，三組HE病理評(píng)分有統(tǒng)計(jì)學(xué)差異。 2.3治療20天EAE組炎性細(xì)胞和“血管袖套”減少，丹參酮組及馬索羅酚組炎性細(xì)胞更少，“血管袖套”消失，三組HE病理評(píng)分有統(tǒng)計(jì)學(xué)差異。 3LFB染色： 三組小鼠在治療前，髓鞘并無(wú)明顯的脫失部位，但仍能觀察到脊髓白質(zhì)內(nèi)部分髓鞘變薄，顏色變淺。EAE組在發(fā)病高峰期可觀察到脊髓內(nèi)存在大小不等的片狀脫髓鞘區(qū)域，病變大多表現(xiàn)在前后角周圍內(nèi)的白質(zhì)，藍(lán)色髓鞘部位減少，顏色變淡，甚至出現(xiàn)了大片狀氣球樣空泡變性的髓鞘脫失區(qū)域，髓鞘脫失部位常伴隨著大片炎癥細(xì)胞浸潤(rùn)，表明髓鞘脫失和炎癥浸潤(rùn)有一定關(guān)系，在治療20d后較高峰期脫髓鞘區(qū)域內(nèi)病變無(wú)明顯改變；而丹參酮組及馬索羅酚組在治療10d后可見部分髓鞘變薄，顏色變淺，存在一些小的髓鞘脫失區(qū)，但沒有形成大片狀髓鞘脫失區(qū)域，病灶散在成點(diǎn)灶狀，治療20d較治療前及治療10d髓鞘變粗，藍(lán)色髓鞘區(qū)域有所增加，三組髓鞘病理評(píng)分在治療10d及治療20d均具有統(tǒng)計(jì)學(xué)差異。 4．Bielschowsky's神經(jīng)軸索染色: 4.1治療前：三組小鼠均已存在軸索損傷，損傷的軸索表現(xiàn)為形狀不規(guī)則、排列紊亂、腫脹扭曲。 4.2治療10天：EAE組處于發(fā)病高峰期，軸索損傷更為明顯，病灶成大片狀，形態(tài)更加不規(guī)則，直徑大小不一，軸索斷裂，斷端形成卵圓小體，甚至出現(xiàn)軸索的消失，部位與炎性細(xì)胞浸潤(rùn)及髓鞘的脫失具有一致性，并且與疾病的嚴(yán)重程度相關(guān)，丹參酮組及馬索羅酚組軸索也存在損失，但范圍及程度與EAE組相比較輕。 4.3治療20天：EAE組在治療20d后與治療10天相比，無(wú)明顯變化，丹參酮組及馬索羅酚組損傷較治療前及治療10d較輕。 結(jié)論： 1馬索羅酚和丹參酮ⅡA作為干預(yù)藥物能改善EAE小鼠的臨床癥狀，為臨床治療多發(fā)性硬化奠定實(shí)驗(yàn)基礎(chǔ)。 2馬索羅酚和丹參酮ⅡA對(duì)EAE小鼠在抗炎、髓鞘脫失、軸索損傷方面具有治療作用
[Abstract]:Objective : To study the clinical manifestation and pathological characteristics of the central nervous system inating disease ( CNS ) . The results showed that the etiology of the MS was related to the genetic factors and the environmental factors . The results showed that the etiology of the MS was related to the genetic factors and the environmental factors .

Methods : Forty - five mice of 8 - 12 weeks old and healthy C57BL / 6 female mice were randomly divided into three groups , i.e . , the rats were randomly divided into three groups , i.e . , the rats were injected with 5 % DMSO and 5 mg / ml .

Results :

1 Animal Clinical Symptoms and Score Observations :

1.1 Clinical symptoms :

Forty - five mice were randomly divided into two hind limbs .

1.2 Observation of clinical score :

The scores of neurological function were higher in all groups than in tanshinonegroup ( P > 0.05 ) .

2HE staining :

2.1 Before treatment , three groups were scattered in inflammatory cells , mostly in the vicinity of white matter in the spinal cord .

2.2 During the treatment of 10 days , the infiltration of large flam - shaped inflammatory cells was seen and even the phenomenon of " blood vessel cuff " was formed , that is , there were inflammatory cells around the small vessels around the spinal cord , especially in the periphery of the small vein . Most of them were lymphocytes . Most of the lesions were in the anterior horn white matter , the gray white matter junction and the anterior median fissure blood vessel , the tanshinone group and the horse sorool group " blood vessel cuff " were formed less , the inflammatory cells were mainly concentrated around the small blood vessels , and the HE pathological scores were statistically different .

2.3 There were fewer inflammatory cells and " blood vessel cuff " in the 20 days of treatment , and there were fewer inflammatory cells in the group of tanshinone and mansorool , and the " blood vessel cuff " disappeared , and the HE pathological scores of the three groups were statistically different .

3LFB staining :

There was no significant loss of myelin sheath in the spinal cord before and after treatment .
After 10 days of treatment , the partial myelin sheath became thinner and the color became lighter . There were some small myelinated depigmentation areas . However , there was no area of large flake myelination , and the lesions were scattered in the focal point . The lesions were treated at 20 days before and after treatment . The myelin sheath became thicker , the blue myelin area increased , and the three groups of myelinated pathological scores had statistical difference in the treatment of 10d and 20d .

4 . Bielschowsky ' s neuroaxonal staining :

4.1 Prior to treatment : all three groups of mice had axonal injury , and the injured axons were characterized by irregular shape , disorder of arrangement , and swelling and distortion .

4.2 Treatment for 10 days : the group was in the peak period , axonal injury was more obvious , the focus became larger , the shape was more irregular , the diameter was small , the axon was broken , the broken end formed the small body of the oval , and even the disappearance of the axon appeared , the site was related to the severity of the disease , there was also the loss of the axonal injury of the tanshinone group and the mansorool group , but the extent and extent were lighter than that of the experimental group .

4.3 Treatment for 20 days : After 20 days of treatment , there was no significant change in the treatment period compared with 10 days after treatment .

Conclusion :

Massorool and tanshinone 鈪 can improve the clinical symptoms and lay the foundation for clinical treatment of multiple sclerosis .

2 - Massorool and tanshinone 鈪 have therapeutic effects on anti - inflammatory , myelinating and axonal injury in mice .

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R965;R744.51

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 張彥博;馬索羅酚（NDGA）對(duì)實(shí)驗(yàn)性自身免疫性腦脊髓炎小鼠炎癥因子表達(dá)的影響[D];河北醫(yī)科大學(xué);2015年

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