本文選題：硒代胱氨酸 + 膠質(zhì)瘤��； 參考：《山東大學(xué)》2016年博士論文

【摘要】：人腦膠質(zhì)瘤來源于神經(jīng)上皮組織,是顱內(nèi)原發(fā)性腫瘤中最常見的類型,近年來呈患病率增高及患者年輕化的趨勢。腦膠質(zhì)瘤侵襲性強(qiáng),常通過侵襲血管壁及膠質(zhì)細(xì)胞間的連接來浸潤、壓迫和破壞腦組織。由于其與正常腦組織分界不清,手術(shù)難以徹底切除,術(shù)后極易復(fù)發(fā)。而其對放療及化療的敏感性均欠佳,故患者預(yù)后差,死亡率高。目前腦膠質(zhì)瘤的治療仍然是一個(gè)難題,對人類生命健康造成嚴(yán)重威脅,因此,尋找更好的抗膠質(zhì)瘤藥物具有十分重要的意義。糖尿病最易并發(fā)腦血管病變,持續(xù)的高血糖引發(fā)嚴(yán)重的神經(jīng)毒性,使患者神經(jīng)功能受損,預(yù)后不良。據(jù)統(tǒng)計(jì),糖尿病合并腦缺血患者的病殘率、復(fù)發(fā)率及死亡率均顯著高于非糖尿病腦缺血患者,給社會和家庭帶來沉重負(fù)擔(dān),已成為目前嚴(yán)重危害公民健康和生命的重大公共衛(wèi)生問題。此外,即使無糖尿病,缺血性腦卒中后患者血糖水平也常常會升高,加重腦損傷�！吨袊毙匀毖阅X卒中診治指南2014))指出：“約40%的患者存在卒中后高血糖,對預(yù)后不利�！币虼�,除了降血糖外,迫切需要有效措施抑制高血糖毒性,改善神經(jīng)病損,促進(jìn)神經(jīng)功能恢復(fù),進(jìn)而提高臨床治療效果。腦卒中是人類災(zāi)難性疾病,具有高發(fā)病率、高死亡率和高致殘率等特點(diǎn),是世界范圍內(nèi)死亡和長期致殘的三大原因之一,同時(shí)也是世界上單病種導(dǎo)致成人后天功能障礙的首位原因。2008年第三次全國死因調(diào)查表明,腦卒中已躍居為我國人口死亡原因的第一位；在各種原因所致死亡中,我國死于腦卒中的構(gòu)成比是歐美發(fā)達(dá)國家的4-5倍。腦缺血再灌注損傷機(jī)制復(fù)雜,氧化還原平衡的破壞是其重要的病理特征。因此,探索高效的抗氧化神經(jīng)保護(hù)劑對腦缺血再灌注損傷的改善及治療具有重要的研究意義和臨床價(jià)值。硒是人體必需的微量元素,對人體生命活動必不可少,可發(fā)揮抗氧化、抗腫瘤、提高免疫力、拮抗重金屬等多種作用。已證實(shí),硒對維持正常神經(jīng)功能發(fā)揮有重要作用；而硒缺乏可影響神經(jīng)認(rèn)知功能及導(dǎo)致神經(jīng)退行性變,如阿爾茨海默病、帕金森病。大量臨床及動物研究證據(jù)顯示,高劑量的硒能加劇氧化應(yīng)激,通過誘導(dǎo)活性氧族物質(zhì)(reactive oxygen species, ROS)的累積誘導(dǎo)氧化損傷,發(fā)揮促凋亡的功效；而低劑量的硒則具有拮抗氧化應(yīng)激、抑制細(xì)胞凋亡的作用。故人們常常采用高劑量硒作為腫瘤化療制劑,誘導(dǎo)腫瘤細(xì)胞周期阻滯,甚至凋亡,以達(dá)到抑癌、抗癌目的；而采用低劑量硒作為細(xì)胞保護(hù)劑,對抗機(jī)體內(nèi)的氧化應(yīng)激損傷。硒代胱氨酸(selenocystine, SeC)是自然可獲取的小分子有機(jī)硒化合物,被稱為人類第21個(gè)必需氨基酸,因其多重藥理學(xué)功效得到廣泛應(yīng)用,尤其是其對氧化還原通路的調(diào)節(jié)引起眾多研究者的高度關(guān)注。3,3’-二硒二丙酸(3,3'-diselenodipropionic acid, DSePA)是SeC的衍生物,性質(zhì)穩(wěn)定、安全性高,具有高效的抗氧化活性,廣泛用于抗氧化研究,在體外和體內(nèi)實(shí)驗(yàn)中對急性神經(jīng)毒性和慢性神經(jīng)退行性病變均表現(xiàn)出保護(hù)效果。據(jù)報(bào)道,硒缺乏可大大增加機(jī)體患癌風(fēng)險(xiǎn),而補(bǔ)充硒可降低某些癌癥的發(fā)病率。SeC具有廣譜的抗腫瘤活性,可通過誘導(dǎo)凋亡在體內(nèi)、外抑制多種人類腫瘤細(xì)胞生長,如肝癌、肺腺癌、乳腺癌及黑色素細(xì)胞瘤,具有潛在的臨床應(yīng)用價(jià)值。近幾年研究還發(fā)現(xiàn)：硒化合物聯(lián)合抗腫瘤藥物一起使用,可增強(qiáng)抗腫瘤藥物的敏感性,即硒化合物可作為腫瘤化療的增敏劑。然而,SeC是否對腦膠質(zhì)瘤細(xì)胞的生長具有抑制作用,其機(jī)制如何,目前尚未見有報(bào)道。大腦屬于高耗氧器官,神經(jīng)元對葡萄糖的高消耗必然伴隨自由基的大量產(chǎn)生；而高血糖會破壞細(xì)胞內(nèi)的抗氧化酶系,削弱其清除能力,使大量自由基無法被及時(shí)清除。持續(xù)高血糖可影響和破壞線粒體內(nèi)膜,啟動線粒體膜電位耗散,導(dǎo)致大量自由基外泄。過多自由基的沉積,會氧化質(zhì)膜,破壞神經(jīng)突觸,擾亂神經(jīng)元之間的連接,甚至導(dǎo)致神經(jīng)元凋亡。實(shí)驗(yàn)證實(shí),拮抗高血糖誘導(dǎo)的氧化應(yīng)激損傷可明顯減弱神經(jīng)損傷／毒性,改善神經(jīng)病變。然而,有機(jī)硒是否對高糖誘導(dǎo)的神經(jīng)毒性有拮抗作用,其機(jī)制如何,目前尚未見有報(bào)道。急性缺血性腦卒中發(fā)作中,腦缺血再灌注損傷的病理機(jī)制復(fù)雜,氧化還原平衡的破壞是其重要的病理特征。氧化應(yīng)激通過ROS超載導(dǎo)致脂質(zhì)過氧化和DNA損傷,在缺血再灌注腦損傷中發(fā)揮關(guān)鍵作用。因此,探索高效的抗氧化神經(jīng)保護(hù)劑對腦缺血再灌注損傷的改善及治療具有重要的研究意義和臨床價(jià)值。而硒對腦缺血再灌注的直接保護(hù)作用,尚未見有研究報(bào)道。故本課題開展了在神經(jīng)系統(tǒng)疾病方面,SeC及其衍生物DSePA的作用研究,探討有機(jī)硒對腦膠質(zhì)瘤、高糖神經(jīng)毒性、缺血性腦卒中的作用效果及機(jī)理。首先,本研究選取了人腦膠質(zhì)瘤細(xì)胞系U251、U87為研究對象,研究SeC對U87和U251細(xì)胞周期的影響,同時(shí)從影響腫瘤信號轉(zhuǎn)導(dǎo)通路的重要信號蛋白MAPKs和AKT入手,分析其可能的作用機(jī)制,為人腦膠質(zhì)瘤的治療提供實(shí)驗(yàn)依據(jù)。其次,本研究選取PC12細(xì)胞作為神經(jīng)細(xì)胞模型,選用DSePA對高糖誘導(dǎo)的毒性損傷進(jìn)行干預(yù),探討其對高糖神經(jīng)毒性的拮抗作用。最后,本研究構(gòu)建了短暫性局灶性腦缺血再灌注(transient focal cerebral ischemia/reperfusion injury, tFCI/R)損傷小鼠模型,以SeC進(jìn)行干預(yù),探討其對缺血再灌注損傷的保護(hù)作用及機(jī)制。目的1.探究SeC對U251、U87兩種人腦膠質(zhì)瘤細(xì)胞系的生長抑制效果,并探討其潛在的分子機(jī)制。2.探究DSePA在PC12細(xì)胞中對高糖誘導(dǎo)神經(jīng)毒性的拮抗效果及機(jī)制。3.探究SeC對tFCI/R損傷小鼠的保護(hù)功效及機(jī)制。方法1.取對數(shù)生長期U251和U87細(xì)胞體外培養(yǎng),待細(xì)胞貼壁后隨機(jī)分為正常對照組、各劑量SeC用藥組。分別以5、10、20μM的SeC處理24 h或48 h。使用相差顯微鏡觀察各組細(xì)胞形態(tài)學(xué)變化；采用四甲基偶氮唑藍(lán)(MTT)比色法測定細(xì)胞生存率,了解SeC對U251和U87細(xì)胞生長的抑制；應(yīng)用流式細(xì)胞術(shù)分析SeC對U251和U87細(xì)胞周期分布的影響；TUNEL-DAPI法觀察SeC對U251細(xì)胞DNA的損傷情況；采用DCFH-DA探針和超氧陰離子試劑盒檢測ROS和超氧陰離子的產(chǎn)生；Western blotting檢測SeC對U251中細(xì)胞周期調(diào)控相關(guān)蛋白Cyclin A,細(xì)胞凋亡相關(guān)蛋白p21、p53及細(xì)胞內(nèi)相關(guān)信號通路的蛋白水平(MAPKs及AKT)的影響。同時(shí),應(yīng)用SeC聯(lián)合多種通路蛋白抑制劑研究其對細(xì)胞周期分布的影響。2.取對數(shù)生長期PC12細(xì)胞體外培養(yǎng),待細(xì)胞貼壁后隨機(jī)分為正常對照組、DSePA用藥組,100 mM葡萄糖處理48 h制作高糖損傷細(xì)胞模型,加入DSePA進(jìn)行干預(yù)。采用流式細(xì)胞分析技術(shù)及TUNEL-DAPI共染色觀察細(xì)胞凋亡情況;檢測caspase-3/-8/-9活性,結(jié)合Western blotting結(jié)果確定凋亡路徑；通過JC-1探針評價(jià)線粒體膜電位,Mito-tracter與DAPI共染色檢測線粒體結(jié)構(gòu)改變,MitoSOX染料檢測超氧陰離子；采用DCFH-DA檢測細(xì)胞內(nèi)ROS水平,并加入ROS清除劑進(jìn)一步證實(shí)氧化損傷的作用；使用Western blotting法檢測相關(guān)蛋白表達(dá)。3.手術(shù)制作tFCI/R小鼠模型,通過多普勒血流監(jiān)測和神經(jīng)功能缺損評分篩選手術(shù)合格的小鼠,隨機(jī)分至SeC治療組(SeC)和溶媒對照組(Vehicle),同時(shí)設(shè)假手術(shù)對照組(Sham,小鼠經(jīng)歷手術(shù)全程而不進(jìn)行大腦中動脈阻塞)。SeC組小鼠給予術(shù)后連續(xù)3日,每日1次腹腔注射SeC溶液(2 mg/30 g),Vehicle組和Sham組小鼠給予同樣方式注射等量生理鹽水。于術(shù)后24、48、72 h各進(jìn)行一次神經(jīng)功能缺損評分及神經(jīng)行為學(xué)測試；手術(shù)3天后行TTC染色測腦梗死體積,干-濕重法測腦水腫程度變化,免疫熒光染色觀察AQP4表達(dá)情況,TUNEL-DAPI染色觀察神經(jīng)元凋亡情況,Western blotting檢測活性caspase-3和活性caspase-9表達(dá)。結(jié)果1.MTT結(jié)果顯示,SeC時(shí)間/劑量依賴性地抑制了U251和U87細(xì)胞的生長,鏡下觀察可見細(xì)胞變圓,突觸減少,細(xì)胞數(shù)目明顯減少,與正常細(xì)胞相比,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；流式細(xì)胞術(shù)結(jié)果表明SeC處理誘導(dǎo)了明顯的U251和U87細(xì)胞S期細(xì)胞周期阻滯,這與cyclin A蛋白表達(dá)下調(diào)相一致；TUNEL-DAPI分析和ROS檢測結(jié)果顯示,SeC處理導(dǎo)致了ROS升高誘導(dǎo)的U251細(xì)胞DNA損傷；Western blotting結(jié)果表明,SeC處理顯著上調(diào)了DNA損傷標(biāo)志物p21和p53的表達(dá),并引起pJNK、p38、pERK的表達(dá)上調(diào)和pAKT的表達(dá)下降。2. DSePA的預(yù)處理可有效減弱高糖對PC12細(xì)胞的毒性作用。流式細(xì)胞技術(shù)檢測到,DSePA預(yù)處理可顯著抑制高糖誘導(dǎo)的PC12細(xì)胞凋亡,表現(xiàn)為sub-G1峰的降低；熒光染色技術(shù)結(jié)果表明,DSePA預(yù)處理可有效抑制高糖誘導(dǎo)的染色質(zhì)的斷裂和核濃縮。caspases活性檢測顯示DSePA抑制了高糖誘導(dǎo)的caspases活性,Western blotting術(shù)從蛋白水平檢測發(fā)現(xiàn),高糖誘導(dǎo)的PARP切割和caspase-3/-7/-9激活被DSePA預(yù)處理顯著抑制；線粒體膜電位及結(jié)構(gòu)檢測證實(shí),DSePA阻斷了高糖誘導(dǎo)的線粒體損傷。DSePA抑制了PC12細(xì)胞中高糖誘導(dǎo)的超氧陰離子產(chǎn)生和ROS累積。3. tFCI/R術(shù)后24、48、72 h神經(jīng)功能缺損評分與神經(jīng)行為學(xué)測試表明,隨時(shí)間延長,所有小鼠神經(jīng)功能損傷及感覺運(yùn)動功能障礙均得到一定程度的改善,而SeC治療組改善顯著,與Vehicle組相比有統(tǒng)計(jì)學(xué)差異；SeC治療組在手術(shù)3天后腦梗死體積和水腫程度上較Vehicle組均有明顯降低,AQP4的表達(dá)比Vehicle組顯著降低,表明SeC明顯緩解了腦組織的梗死和水腫；TUNEL-DAPI染色、Western blotting結(jié)果表明,SeC處理顯著下調(diào)了caspase-3和PARP的裂解,從而抑制了神經(jīng)元凋亡。結(jié)論1.SeC劑量/時(shí)間依賴地抑制人腦膠質(zhì)瘤細(xì)胞的增殖,其分子機(jī)制為通過調(diào)節(jié)MAPKs和AKT信號通路誘導(dǎo)人腦膠質(zhì)瘤細(xì)胞S期阻滯。2.DSePA可通過抑制氧化應(yīng)激誘發(fā)的細(xì)胞凋亡,拮抗高糖神經(jīng)毒性,有望成為拮抗高糖誘導(dǎo)神經(jīng)系統(tǒng)疾病的高效策略。3.腹腔注射SeC對小鼠腦缺血再灌注損傷確有保護(hù)功效,主要是通過抑制水腫,抵抗氧化作用誘導(dǎo)的神經(jīng)元凋亡。
[Abstract]:Brain glioma is the most common type in the primary tumor of the brain . It is the most common type in the primary tumor of the brain . It is very important for patients with cerebral glioma .
Selenium is essential for human life and plays an important role in maintaining normal nerve function . Selenium is essential for human life and can exert antioxidant , anti - tumor , immunity enhancing , and heavy metals .
However , selenium deficiency can affect neurocognitive function and cause neurodegeneration , such as Alzheimer ' s disease and Parkinson ' s disease . High - dose selenium can aggravate oxidative stress , induce oxidative damage by inducing accumulation of reactive oxygen species ( ROS ) , and exert the effect of promoting apoptosis .
while the low - dose selenium has the effect of inhibiting oxidative stress and inhibiting apoptosis , so that people often adopt high - dose selenium as a tumor chemotherapeutic agent , induce tumor cell cycle arrest and even apoptosis , so as to achieve the purpose of inhibiting cancer and resisting cancer ;
SeC is a naturally - available small molecule organic selenium compound , which is called the 21st essential amino acid of human body .
In this study , we have studied the effects of SeC on brain glioma , hyperglycotoxicity and ischemic stroke , and discussed the effect and mechanism of SeC on brain glioma , high glucose neurotoxicity and ischemic stroke .
The survival rate of cells was determined by MTT assay , and the inhibition of SeC on U251 and U87 cell growth was investigated .
Effect of SeC on Cell Cycle Distribution of U251 and U87 by Flow Cytometry
The DNA damage of U251 cells was observed by TUNEL - DAPI method .
the generation of ROS and superoxide anion is detected by adopting a DCFH - DA probe and a superoxide anion kit ;
The effects of SeC on cell cycle distribution in U251 were studied by Western blotting . The effects of SeC on cell cycle distribution were studied . At the same time , the effects of SeC combined with various pathway protein inhibitors on cell cycle distribution were studied .
The mitochondrial membrane potential was evaluated by JC - 1 probe , Mito - tracter and DAPI staining were used to detect mitochondrial structure changes .
DCFH - DA was used to detect ROS level in cells , and ROS scavenger was added to further confirm the effect of oxidative damage .
Western blotting was used to detect the expression of related protein . 3 . The tFCI / R mouse model was produced by operation , and the eligible mice were selected by Doppler blood flow monitoring and neurological deficit scoring . The mice were randomly divided into the SeC treatment group ( SeC ) and the vehicle control group ( Vehicle ) , and the sham operation control group ( Sham , the mice underwent the whole course of surgery without the middle cerebral artery occlusion ) . SeC solution ( 2 mg / 30 g ) , Vehicle group and Sham group mice were injected with the same amount of physiological saline at 24 , 48 and 72 hours after the administration of SeC solution ( 2 mg / 30 g ) . The neurological deficit score and neurobehavioral test were performed at 24 , 48 and 72 hours after operation .
Results 1 . The results showed that SeC time / dose dependently inhibited the growth of U251 and U87 cells . The results showed that SeC time / dose dependently inhibited the growth of U251 and U87 cells . The results showed that SeC time / dose dependently inhibited the growth of U251 and U87 cells .
Flow cytometry showed that SeC treatment induced cell cycle arrest of U251 and U87 cells , which was consistent with the downregulation of cyclin A protein .
TUNEL - DAPI analysis and ROS test showed that SeC treatment led to the DNA damage of U251 cells induced by the increase of ROS .
Western blotting showed that SeC treatment significantly raised the expression of p21 and p53 in DNA damage markers , and resulted in the up - regulation of p38 and pERK and decreased expression of pERK . The pretreatment of DSePA could reduce the toxicity of high glucose to PC12 cells . Flow cytometry showed that DSePA pretreatment could significantly inhibit the apoptosis of PC12 cells induced by high glucose , and showed a decrease of sub - G1 peak .
The results of fluorescence staining showed that DSePA pretreatment could effectively inhibit the cleavage and nuclear concentration of high glucose - induced chromatin . caspases activity assay showed that DSePA inhibited the activity of high glucose - induced caspases . Western blotting showed that high glucose - induced cleavage and caspase - 3 / -7 / -9 activation were significantly inhibited by DSePA pretreatment .
DSePA inhibited high glucose induced superoxide anion production and ROS accumulation in PC12 cells . DSePA inhibited high glucose induced superoxide anion production and ROS accumulation in PC12 cells .
There was a significant decrease in cerebral infarction volume and degree of edema in the SeC treatment group after 3 days of operation . The expression of AQP4 was significantly lower than that in Vehicle group , indicating that SeC significantly alleviated the infarction and edema of brain tissue ;
Conclusion 1 . SeC dose / time can inhibit the proliferation of human glioma cells . Conclusion 1 . SeC dose / time can inhibit the proliferation of human glioma cells .

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R739.41
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本文編號：1822290