發(fā)布時(shí)間：2018-05-15 13:42

  本文選題：活性氧自由基 + 阿爾茲海默病　； 參考：《第四軍醫(yī)大學(xué)》2013年博士論文

【摘要】：目的: 阿爾茲海默病(Alzheimer's disease, AD)是老年期癡呆最常見(jiàn)的類型。認(rèn)知能力的逐漸下降和淀粉樣斑塊及神經(jīng)原纖維纏結(jié)的出現(xiàn)是其典型的臨床病理特征。最終結(jié)果會(huì)導(dǎo)致腦功能的異常及神經(jīng)元凋亡。隨著我國(guó)人口老齡化的日益嚴(yán)重，AD越來(lái)越成為一個(gè)嚴(yán)重的社會(huì)問(wèn)題。研究表明AD神經(jīng)退行性病變過(guò)程中，氧化應(yīng)激是除年齡外另一個(gè)重要因素。大量的研究表明DNA、RNA、脂質(zhì)和蛋白質(zhì)氧化程度在AD病變過(guò)程中不斷升高，直至出現(xiàn)輕度認(rèn)知損傷，這些都說(shuō)明氧化應(yīng)激出現(xiàn)在AD的早期階段，即發(fā)生在淀粉樣斑塊和神經(jīng)原纖維纏結(jié)之前出現(xiàn)。盡管在AD的治療研究中有很多針對(duì)于對(duì)抗氧化應(yīng)激介導(dǎo)損傷的神經(jīng)保護(hù)策略，但是臨床結(jié)果積極有效的只有很少一部分。 氮氧自由基（Nitroxide radicals, NRs）作為一類含有自旋單電子的穩(wěn)定的自由基化合物。早期被用作自旋示蹤劑。近來(lái)研究表明NRs還具有其特殊的生物學(xué)活性，一些NRs具有抗腫瘤、輻射和缺血再灌注損傷等功能。它與有害自由基反應(yīng)通過(guò)“催化劑量”方式進(jìn)行，只需很少的劑量即可發(fā)揮高效、強(qiáng)效和長(zhǎng)效的抗氧化活性。例如，NRs具有擬SOD的作用。它可以通過(guò)電子轉(zhuǎn)移的方式參與到細(xì)胞內(nèi)的線粒體的呼吸鏈?zhǔn)椒磻?yīng)，非�？焖俚那宄蹶庪x子自由基O2.-。正是由于NRs以催化方式分解不斷產(chǎn)生的有害自由基，反應(yīng)過(guò)程中本身不會(huì)被消耗，可以循環(huán)再生利用，這也是其它自由基清除劑所無(wú)法比擬的。同時(shí)因其特有的自旋示蹤功能，可通過(guò)電子順磁共振或核磁共振成像實(shí)時(shí)了解其在組織中的分布及變化反應(yīng)。 正是由于NRs這些獨(dú)特的性質(zhì)，我們推測(cè)其在AD的發(fā)病過(guò)程中會(huì)發(fā)揮很好的抗氧化的能力，起到預(yù)防、治療甚至是診斷AD發(fā)生的作用。因此，有望將NRs制備成新型的應(yīng)用于神經(jīng)退行性疾病的抗氧化應(yīng)激損傷的藥物。 方法： 在本實(shí)驗(yàn)中，我們采用體外抗氧化模型，Aβ1-42誘導(dǎo)的細(xì)胞毒性損傷模型以及APP/PS1雙轉(zhuǎn)基因AD鼠模型，來(lái)評(píng)價(jià)NRs的抗氧化能力及對(duì)AD的保護(hù)作用。 體外抗氧化實(shí)驗(yàn)中，我們分別采用光澤精誘導(dǎo)的發(fā)光體系、CHP誘導(dǎo)的脂質(zhì)過(guò)氧化模型和F2-異前列素免疫試劑盒來(lái)評(píng)價(jià)NRs清除超氧陰離子和抑制脂質(zhì)過(guò)氧化的能力； 體外培養(yǎng)原代皮層神經(jīng)元模型中，在培養(yǎng)到第10天的時(shí)候，分別給予10μM的姜黃素、Tempol和L-NNNBP，孵育24小時(shí)后，用25μM的寡聚態(tài)Aβ1-42處理12小時(shí)。換用原培養(yǎng)液繼續(xù)培養(yǎng)24小時(shí)后收集細(xì)胞。分別用CCK-8試劑盒檢測(cè)細(xì)胞的存活率；用Tunel試劑盒檢測(cè)細(xì)胞的凋亡；用3-NT的ELISA試劑盒測(cè)定組織的硝化應(yīng)激水平；用陽(yáng)離子熒光染料-四甲基羅丹明乙酯檢測(cè)線粒體的膜電位變化；用細(xì)胞的免疫熒光觀察激活的Caspase-3的變化； 最后采用APP/PS-1雙轉(zhuǎn)基因AD小鼠來(lái)評(píng)價(jià)NRs的自由基清除能力和對(duì)AD的保護(hù)作用。實(shí)驗(yàn)分為5組：姜黃素治療組、Tempol治療組、L-NNNBP治療組、WT組和TG組。藥物用飲用水溶解，終濃度均為1mM，WT組和TG組給予正常飲水。在小鼠6周時(shí)給予藥物處理，連續(xù)給藥1個(gè)月。用Morris水迷宮實(shí)驗(yàn)評(píng)價(jià)AD小鼠空間學(xué)習(xí)記憶能力；用剛果紅染色方法觀察Aβ斑塊；用蛋白免疫印跡方法觀察磷酸化Tau和GFAP蛋白含量變化；采用免疫組化方法觀察星形膠質(zhì)細(xì)胞的激活。 結(jié)果： 體外抗氧化實(shí)驗(yàn)中，我們發(fā)現(xiàn)L-NNNBP較姜黃素和Tempol能顯著減少超氧陰離子和脂質(zhì)過(guò)氧化水平(#P0.05，##P0.01)。 培養(yǎng)皮層神經(jīng)元模型中，不同劑量的L-NNNBP對(duì)Aβ1-42誘導(dǎo)的細(xì)胞毒性都有保護(hù)作用(*P0.05，**P0.01)，且是濃度依賴的關(guān)系。單獨(dú)給予L-NNNBP，沒(méi)有對(duì)細(xì)胞產(chǎn)生毒性作用。而Tempol的三種劑量都沒(méi)有觀察到神經(jīng)元的保護(hù)作用；姜黃素只有在高劑量（10μM）時(shí)有細(xì)胞保護(hù)作用。Tunel法檢測(cè)細(xì)胞凋亡的實(shí)驗(yàn)中，相對(duì)于姜黃素（42.9%±3.1%）和Tempol（51.1%±1.1%），L-NNNBP高劑量（10μM）處理組，神經(jīng)元的凋亡顯著降低（22.7%±2.6%，*P0.05,**P0.01,##P0.01）。細(xì)胞免疫熒光結(jié)果說(shuō)明L-NNNBP的這種抗凋亡的作用是通過(guò)降低激活型Caspase-3的表達(dá)引起的。Aβ誘導(dǎo)的氧化應(yīng)激損傷也會(huì)造成線粒體功能障礙，影響線粒體正常膜電位，使其去極化。L-NNNBP能顯著降低TMRM+的密度，阻止線粒體膜的去極化，保護(hù)線粒體的正常功能，，且其作用強(qiáng)度顯著高于姜黃素和Tempol（#P0.05）。除了氧化應(yīng)激，硝化應(yīng)激也是自由基產(chǎn)生的主要來(lái)源。L-NNNBP三個(gè)濃度都降低了3-NT的含量（**P0.01），且抑制率顯著高于姜黃素和Tempol（#P0.05）。 Aβ斑塊的沉積是AD的主要病理癥狀之一。在APP/PS1雙轉(zhuǎn)基因AD鼠模型中，6個(gè)月大的APP/PS-1小鼠給予L-NNNBP(1mM)1個(gè)月后，海馬和皮層的Aβ斑塊的沉積都顯著減少，并且效果強(qiáng)于姜黃素和Tempol（##P0.05）。除了Aβ斑塊沉積，AD的另一個(gè)病理特征就是Tau蛋白異常過(guò)度磷酸化后引起的神經(jīng)原纖維纏結(jié)。APP/PS1小鼠Tau蛋白的Thr205和Ser235兩個(gè)位點(diǎn)的磷酸化程度顯著升高。相對(duì)于WT組和其它藥物處理組，L-NNNBP顯著的降低了Tau蛋白在兩個(gè)位點(diǎn)的磷酸化程度（**P0.01，#P0.05，##P0.05）。星形膠質(zhì)細(xì)胞的免疫組化和免疫蛋白印跡結(jié)果說(shuō)明其在AD發(fā)病的早期就被激活，有可能進(jìn)一步誘導(dǎo)β-淀粉樣蛋白沉積加速和神經(jīng)纖維纏結(jié)形成。而L-NNNBP在發(fā)揮抗氧化功效的同時(shí)抑制了星形膠質(zhì)細(xì)胞的激活。Morris水迷宮的行為學(xué)結(jié)果也說(shuō)明APP/PS1小鼠空間學(xué)習(xí)和記憶能力受到損傷，在給予L-NNNBP治療后有顯著改善，并且效果強(qiáng)于姜黃素和Tempol（**P0.01,##P0.01）。 結(jié)論： 本研究證明，無(wú)論是在氧化損傷的體外模型，還是Aβ1-42誘導(dǎo)的細(xì)胞毒性損傷模型，或是APP/PS1雙轉(zhuǎn)基因AD鼠模型中，新型的手性氮氧自由基L-NNNBP都表現(xiàn)出了令人興奮的結(jié)果。它能清除超氧陰離子、抑制脂質(zhì)過(guò)氧化、減輕Aβ1-42誘導(dǎo)的細(xì)胞毒性損傷、抗凋亡、降低氧化或硝化應(yīng)激損傷、減少Aβ斑塊沉積、降低Tau蛋白磷酸化水平、抑制星形膠質(zhì)細(xì)胞激活和改善AD鼠的空間學(xué)習(xí)和記憶。總之，L-NNNBP很有可能開(kāi)發(fā)成為臨床AD治療的候選藥物。
[Abstract]:Purpose :

Alzheimer ' s disease ( AD ) is one of the most common types of Alzheimer ' s disease . The gradual decline of cognitive ability and the appearance of amyloid plaques and neurogenesis are typical clinical pathological characteristics . As the aging population of our country becomes more and more serious , AD has become a serious social problem .

NRs is a kind of stable free radical compound containing spin single electrons . It has been used as a spin tracer in the early stage . Recent studies have shown that NRs has its special biological activity , and some NRs have the functions of anti - tumor , radiation and ischemia - reperfusion injury .

Because of the unique nature of NRs , we have speculated that it plays a very good role in preventing , treating and even diagnosing AD during the onset of AD . Therefore , it is hopeful to prepare NRs into a new drug which can be applied to the anti - oxidative stress injury of neurodegenerative diseases .

Method :

In this experiment , we evaluated the anti - oxidation ability of NRs and the protective effect on AD by using in vitro antioxidant model , A尾1 - 42 induced cytotoxicity damage model and APP / PSl double transgenic AD rat model .

In vitro antioxidant experiments , we evaluated the ability of NRs to eliminate superoxide anion and inhibit lipid peroxidation by using the light - emitting system induced by glossiness , the lipid peroxidation model induced by CHP and the F2 - isoalprost immunokit .


In the model of primary cortical neurons cultured in vitro , 10.mu . M of curcumin , Tempol and L - NNNBP were administered to 10.mu . M of curcumin , Tempol and L - NNNBP , respectively , and incubated for 24 hours with 25 & mu ; M of the oligomerized A.beta . 1 - 42 for 12 hours . The cells were collected after continued culturing for 24 hours with the original culture broth . The survival rate of the cells was detected by CCK - 8 kit , respectively ;
The apoptosis of cells was detected by Tunel kit .
The level of nitrification stress was determined by ELISA kit of 3 - NT .
detecting mitochondrial membrane potential changes with cationic fluorescent dye - tetramethylrhodamine ethyl ester ;
The changes of Caspase - 3 were observed by immunofluorescence of cells .


The experiment was divided into five groups : curcumin treatment group , Tempol treatment group , L - NNNBP treatment group , WT group and TG group .
The A 尾 plaques were observed by Congo red staining .
The contents of phosphorylated Tau and GFAP were observed by Western blotting .
Immunohistochemical method was used to observe the activation of astrocytes .

Results :

In vitro antioxidant experiments , we found that L - NNNBP significantly reduced superoxide anion and lipid peroxidation ( P0.05 , P 0.01 ) .

In the cortical neuron model , the cytotoxicity of L - NNNBP induced by different doses of L - NNNBP on A尾1 - 42 was protective ( * P0.05 , ** P0.01 ) .
In the experiment of detecting apoptosis by Tunel method , curcumin ( 42.9 % 鹵 3.1 % ) and Tempol ( 51.1 % 鹵 1.1 % ) , L - NNNBP high dose ( 10 渭M ) treatment group , the apoptosis of neurons decreased significantly ( 22 . 7 % 鹵 2.6 % , * P 0 . 05 , ** P0.01 , P P0.01 ) . L - NNNBP could significantly decrease the density of TMRM + , prevent depolarization of mitochondrial membrane , protect the normal function of mitochondria and protect the normal function of mitochondria , and the effect of L - NNNBP was significantly higher than that of curcumin and Tempol ( # P05 ) .

Compared with WT group and other drug treatment groups , L - NNNBP significantly decreased the phosphorylation of Tau protein in two sites ( ** P0.01 , # P05 , # P0 . 05 ) . Immunohistochemical and Western blot analysis of astrocytes showed that it was activated in the early stage of AD , and it was possible to further induce 尾 - amyloid deposition acceleration and neurofilament entanglement formation . L - NNNBP inhibited astrocytes from activation . The results of Morris water maze also showed that the spatial learning and memory abilities of APP / PSl mice were damaged , and the effect was stronger than curcumin and Tempol ( ** P0.01 , # P0.01 ) .

Conclusion :

In conclusion , the novel chiral nitrogen - oxygen free radical L - NNNBP showed an exciting result either in an in vitro model of oxidative damage , a cytotoxicity damage model induced by A.beta . 1 - 42 , or a novel chiral nitrogen - oxygen free radical L - NNNBP . It can scavenge superoxide anion , inhibit lipid peroxidation , reduce the damage of A尾1 - 42 induced cytotoxicity , reduce A尾 plaque deposition , decrease Tau protein phosphorylation level , inhibit astrocyte activation and improve the spatial learning and memory of AD rats .

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R749.16

【共引文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 李珂;散發(fā)性包涵體肌炎的臨床與病理及蛋白質(zhì)組學(xué)研究[D];中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院;2010年

相關(guān)碩士學(xué)位論文 前1條

1 曹霞飛;白藜蘆醇對(duì)甲醛誘導(dǎo)PC12細(xì)胞氧化損傷的影響[D];南華大學(xué);2010年

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