發(fā)布時(shí)間：2018-07-06 17:40

  本文選題：鐵 + 螯合劑�。� 參考：《第二軍醫(yī)大學(xué)》2012年博士論文

【摘要】：背景 血管性癡呆(vascular dementia,VaD)是各種腦血管病引起的獲得性智能損害和認(rèn)知障礙的綜合征,為一種慢性進(jìn)行性疾病。在導(dǎo)致癡呆的眾多病因中,VaD是僅次于Alzheimer病(Alzheimer's disease, AD)的第二大病因。隨著全球人口的老齡化和腦血管病發(fā)病率的增高,VaD的發(fā)病率也逐年增高,嚴(yán)重危害著人類健康,并給社會(huì)和家庭造成沉重的經(jīng)濟(jì)壓力。就臨床實(shí)際意義而言,VaD是目前唯一可以預(yù)防的癡呆類型,可能較AD更有預(yù)防和治療價(jià)值,早期合理防治VD可以減輕社會(huì)和家庭的沉重負(fù)擔(dān)。正因如此,近年來(lái)VaD逐漸成為人們關(guān)注的熱點(diǎn)。 VaD的發(fā)病機(jī)制可能與以下幾個(gè)因素有關(guān)：中樞膽堿能系統(tǒng)功能障礙、氧自由基生成增加、中樞RNA和蛋白質(zhì)合成減少、局部炎癥反應(yīng)及機(jī)體免疫異常等。由于發(fā)病機(jī)制的多樣化,現(xiàn)有治療手段或藥物(如膽堿酯酶抑制劑、N-甲基-D-天冬氨酸(N-methyl-D-aspartate, NMD A)受體拮抗藥等)均不能完全控制血管性癡呆的所有癥狀或者延緩甚至逆轉(zhuǎn)其進(jìn)程。 最近的研究表明,金屬離子尤其是鐵離子與神經(jīng)退行性疾病的發(fā)生關(guān)系密切[4]。已有動(dòng)物實(shí)驗(yàn)表明,腦組織中鐵離子過(guò)負(fù)荷,與阿爾茨海默病(AD)、帕金森病(PD)等的發(fā)病直接相關(guān)。用鐵離子螯合劑來(lái)治療神經(jīng)退行性疾病的動(dòng)物模型,取得了比較樂觀的效果。 越來(lái)越多的證據(jù)表明AD與VaD經(jīng)常相互伴隨發(fā)病,兩者在病因?qū)W、危險(xiǎn)因素、發(fā)病機(jī)制、病理學(xué)、癥狀學(xué)和疾病轉(zhuǎn)歸等方面都有顯著的重疊。然而鐵離子在血管性癡呆中的作用卻并無(wú)深入探究。 目的 本研究旨在運(yùn)用兩血管法制備血管性癡呆小鼠模型,觀察不同時(shí)間點(diǎn)小鼠行為學(xué)及腦損傷的關(guān)系。并利用鐵螯合劑——甲磺酸去鐵胺對(duì)動(dòng)物模型進(jìn)行干預(yù),觀察其是否對(duì)血管性癡呆模型具有保護(hù)或治療效應(yīng)并初步探討其可能的作用機(jī)制。 第一部分血管性癡呆小鼠模型的建立和評(píng)價(jià) 方法：采用二血管法即雙側(cè)頸總動(dòng)脈完全阻斷1小時(shí)后再灌注的方法造模,并于造模后第3天開始Morris水迷宮訓(xùn)練,第3、7、14、21天測(cè)懸尾實(shí)驗(yàn)靜止不動(dòng)時(shí)間,第7、14、21天測(cè)定位航行實(shí)驗(yàn)上臺(tái)潛伏期和空間探索實(shí)驗(yàn)跨越平臺(tái)次數(shù),第21天測(cè)Open filed實(shí)驗(yàn)休息時(shí)間。第21天觀察小鼠腦組織病理改變(HE染色、尼氏染色)。 結(jié)果：小鼠缺血再灌注組與假手術(shù)組相比,懸尾實(shí)驗(yàn)靜止不動(dòng)時(shí)間、水迷宮定位航行實(shí)驗(yàn)上臺(tái)潛伏期明顯延長(zhǎng),空間探索實(shí)驗(yàn)跨越平臺(tái)次數(shù)明顯減少,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)；但曠場(chǎng)實(shí)驗(yàn)休息時(shí)間,兩組間差異無(wú)明顯統(tǒng)計(jì)學(xué)意義(P0.05)模型組不同時(shí)間點(diǎn)之間比較,隨著再灌注時(shí)間的延長(zhǎng),懸尾靜止不動(dòng)時(shí)間逐漸延長(zhǎng),定位航行上臺(tái)潛伏期逐漸縮短,具有統(tǒng)計(jì)學(xué)意義(P0.05)；但空間探索跨越平臺(tái)次數(shù)的增多未見統(tǒng)計(jì)學(xué)意義(P0.05)。病理HE染色示模型組較假手術(shù)組海馬區(qū)細(xì)胞層次不清、胞體變小。Nissl染色示模型組較假手術(shù)組海馬神經(jīng)元形態(tài)不規(guī)則,著色不均勻,尼氏小體含量明顯減少。 結(jié)論：二血管法制備的血管性癡呆小鼠模型,成模率高,穩(wěn)定性好。 第二部分鐵螯合劑對(duì)血管性癡呆小鼠模型的作用 方法：將在我校實(shí)驗(yàn)動(dòng)物中心購(gòu)得的雄性ICR小鼠160只隨機(jī)分為4組：假手術(shù)組、血管性癡呆模型+生理鹽水對(duì)照組、血管性癡呆模型+甲磺酸去鐵胺(50mg/kg)干預(yù)組及血管性癡呆模型+甲磺酸去鐵胺(100mg/kg)干預(yù)組,每組40只。各組再次隨機(jī)分成4亞組,每組10只,分別標(biāo)記術(shù)后3d、7d、14d、21d。同實(shí)驗(yàn)第一部分制作動(dòng)物模型,再灌注時(shí)給予甲磺酸去鐵胺或等量生理鹽水干預(yù),之后2天相同時(shí)間點(diǎn)再次給予去鐵胺或等量生理鹽水干預(yù)。術(shù)后3、7、14、21天四個(gè)時(shí)間點(diǎn)每組中的各亞組10只小鼠分別進(jìn)行行為學(xué)檢測(cè)、取血及腦組織勻漿行鐵含量檢測(cè)。 結(jié)果：去鐵胺干預(yù)組較生理鹽水空白對(duì)照組術(shù)后7、14、21d的Morris水迷宮定位航行實(shí)驗(yàn)上臺(tái)潛伏期明顯縮短,懸尾不動(dòng)時(shí)間也明顯縮短(P0.05)；而去鐵胺高劑量組與低劑量組相比,上述指標(biāo)差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。水迷宮空間探索實(shí)驗(yàn)在再灌注21d時(shí)去鐵敏不同劑量干預(yù)組較生理鹽水空白對(duì)照組跨越站臺(tái)次數(shù)明顯增多(P0.05)；而7、14d時(shí),各組間無(wú)明顯統(tǒng)計(jì)學(xué)差異(P0.05)。再灌注14天時(shí)曠場(chǎng)實(shí)驗(yàn)小鼠休息時(shí)間,各模型組間無(wú)明顯差異(P0.05) 血清鐵及腦組織勻漿鐵檢測(cè)表明：生理鹽水模型組較假手術(shù)組,四個(gè)時(shí)間點(diǎn)血清及腦組織勻漿鐵含量均明顯增高,有統(tǒng)計(jì)學(xué)意義(P0.05)；去鐵胺干預(yù)組較生理鹽水空白對(duì)照組,術(shù)后3、7、14三個(gè)時(shí)間點(diǎn),血清及腦組織勻漿鐵含量均明顯減少,有統(tǒng)計(jì)學(xué)意義(P0.05) 結(jié)論： 1、鐵螯合劑可改善血管性癡呆小鼠與認(rèn)知有關(guān)的行為學(xué)指標(biāo)； 2、血管性癡呆小鼠的血清和腦組織鐵含量明顯升高； 3、鐵螯合劑干預(yù)血管性癡呆小鼠后,血清和腦組織鐵含量明顯降低,說(shuō)明鐵螯合劑可能是通過(guò)螯合血及腦組織中鐵來(lái)發(fā)揮神經(jīng)保護(hù)作用的。 第三部分鐵螯合劑對(duì)血管性癡呆小鼠模型的神經(jīng)保護(hù)作用機(jī)制探討 方法：實(shí)驗(yàn)動(dòng)物分組和干預(yù)以及血和腦組織標(biāo)本收集同第二部分。小鼠大腦左半球投入4%多聚甲醛后固定48小時(shí)后投入20%蔗糖/PB溶液4℃浸泡至組織沉底。行冰凍切片,選取額葉皮層區(qū)域分別行免疫組化GST-pi染色和MBP染色,記數(shù)前額葉GST-pi陽(yáng)性的少突膠質(zhì)細(xì)胞的數(shù)目以及測(cè)定MBP Density/Area。小鼠大腦右半球腦組織勻漿,分別檢測(cè)腦組織總超氧化物歧化酶活力,抑制羥自由基能力、丙二醛含量。血清測(cè)定乙酰膽堿含量、膽堿酯酶活力。 結(jié)果：去鐵胺干預(yù)組較生理鹽水空白對(duì)照組術(shù)后3、7、14、21d腦組織抑制羥自由基能力增強(qiáng)、丙二醛含量減少、T-SOD活力增強(qiáng),差異均有統(tǒng)計(jì)學(xué)意義(p0.05)血清乙酰膽堿含量、膽堿酯酶活力在各組間無(wú)明顯統(tǒng)計(jì)學(xué)差異(P0.05)。病理結(jié)果提示：?jiǎn)渭兡Ｐ徒M與假手術(shù)組相比,術(shù)后各時(shí)間點(diǎn)額葉皮層下少突膠質(zhì)細(xì)胞數(shù)目明顯減少,前額葉皮層髓鞘堿性蛋白(MBP) Density/Area明顯降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；而去鐵敏干預(yù)組與生理鹽水空白對(duì)照組相比,術(shù)后各時(shí)間點(diǎn)額葉皮層下少突膠質(zhì)細(xì)胞數(shù)目增多、前額葉皮層Density/Area升高,有統(tǒng)計(jì)學(xué)意義(p0.05) 結(jié)論： 1、鐵螯合劑可阻斷氧化應(yīng)激反應(yīng),減少缺血缺氧誘導(dǎo)的血管性癡呆小鼠模型羥自由基和丙二醛生成,增強(qiáng)SOD活力。這種作用能持續(xù)至缺血再灌注21天。 2、鐵螯合劑可以促進(jìn)血管性癡呆小鼠的髓鞘修復(fù),促進(jìn)少突膠質(zhì)細(xì)胞的增生具有膠質(zhì)細(xì)胞保護(hù)作用。這種作用在缺血再灌注3周內(nèi)均比較明顯。
[Abstract]:Background

Vascular dementia ( VaD ) is a syndrome of acquired intellectual impairment and cognitive disorder caused by various cerebrovascular diseases , and is a chronic progressive disease . In many causes of dementia , VaD is the only second major cause of Alzheimer ' s disease ( AD ) . With the aging of global population and the increasing incidence of cerebrovascular disease , VaD is the only type of dementia that can be prevented .

The pathogenesis of VaD may be related to several factors : the dysfunction of central cholinergic system , the increase of oxygen free radicals , the decrease of central RNA and protein synthesis , the local inflammatory response and the abnormal organism immunity , etc . Due to the diversification of the pathogenesis , the existing treatment methods or drugs ( such as cholinesterase inhibitor , N - methyl - D - aspartate ( N - methyl - D - aspartate , nmd A ) receptor antagonist , etc . ) can not completely control all the symptoms of vascular dementia or delay or even reverse its progression .

Recent studies have shown that metal ions , especially iron ions , are closely associated with the onset of neurodegenerative diseases . Animal experiments have shown that the overload of iron ions in brain tissue is directly related to the pathogenesis of Alzheimer ' s disease ( AD ) and Parkinson ' s disease ( PD ) .

There is an increasing number of evidence that AD and VaD are frequently associated with each other , both in etiology , risk factors , pathogenesis , pathology , symptoms , and disease outcomes . However , the role of iron ions in vascular dementia is not deeply explored .

Purpose

The purpose of this study was to use two - vessel method to prepare the model of vascular dementia mice , to observe the relationship between behavior and brain injury in mice at different time points .

Establishment and evaluation of mouse model of vascular dementia in the first part

Methods : A two - vessel method was used to block the total occlusion of bilateral common carotid artery for 1 hour , and the Morris water maze training was started on the 3rd day after the molding . The latency and space exploration experiments on the 3rd , 7th , 14th and 21st day were carried out . On the 7th , 14th and 21st day , the latency and the space exploration experiment were measured across the platform . On the 21st day , the pathological changes of the brain tissues were observed ( HE staining and Nye staining ) .

Results : Compared with the sham operation group , the experimental results showed that the latency period of the experiment was prolonged obviously , and the space exploration experiment significantly decreased the number of stages and the difference was statistically significant ( P0.05 ) .
However , there was no significant difference between the two groups ( P0.05 ) .
However , there was no statistical significance in the spatial exploration of the number of stages across the platform ( P0.05 ) . The pathological HE staining showed that the level of cells in the hippocampus of sham operation group was not clear , and the cell body became smaller . Nissl staining showed that the morphological irregularity , non - uniform coloring and the content of Nissl ' s small body were obviously reduced in the sham operation group .

Conclusion : The model of vascular dementia mice prepared by two - vessel method has high forming rate and good stability .

Effect of the second part of iron chelating agent on the model of vascular dementia mice

Methods : 160 male ICR mice were randomly divided into four groups : sham operation group , vascular dementia model + physiological saline control group , vascular dementia model + methanesulfonic acid deferrioxamine ( 50mg / kg ) intervention group and vascular dementia model + methanesulfonic acid deferrioxamine ( 100mg / kg ) intervention group .

Results : Compared with saline control group , the latency period was significantly shorter than that of saline control group ( 7 , 14 , 21 d ) , and the duration of suspension tail was shortened obviously ( P0.05 ) .
Compared with the low - dose group , there was no significant difference in the above - mentioned indexes ( P0.05 ) .
No significant difference was found between the groups at 7 and 14 days ( P0.05 ) . There was no significant difference between the model groups ( P0.05 ) .

The levels of iron and iron in serum and brain tissue of normal saline group were significantly higher than those in sham operation group and four time points ( P0.05 ) .
The levels of iron in serum and brain tissue were significantly decreased at 3 , 7 and 14 hours after deironing and compared with normal saline control group ( P0.05 ) .

Conclusion :

1 . The iron chelating agent can improve the behavioral indexes of vascular dementia mice and cognition ;


2 . The iron content in serum and brain tissue of vascular dementia mice was significantly increased .


3 . The iron content of serum and brain tissue decreased significantly after the iron chelating agent was used in the treatment of vascular dementia mice , indicating that the iron chelating agent could play a role of neuroprotection by chelating blood and iron in brain tissue .

Neuroprotective effect of the third part of iron chelating agent on the model of vascular dementia mice

Methods : The group and intervention of experimental animals and blood and brain tissue samples were collected in the same second part . After 48 hours , the brain left hemisphere of mice was fixed for 48 hours and then immersed in 20 % sucrose / PB solution at 4 鈩，

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