硒代胱氨酸及其衍生物的抗脑胶质瘤机理及神经保护机制研究

发布时间：2018-04-30 00:36

  本文选题：硒代胱氨酸 + 胶质瘤　； 参考：《山东大学》2016年博士论文

【摘要】：人脑胶质瘤来源于神经上皮组织,是颅内原发性肿瘤中最常见的类型,近年来呈患病率增高及患者年轻化的趋势。脑胶质瘤侵袭性强,常通过侵袭血管壁及胶质细胞间的连接来浸润、压迫和破坏脑组织。由于其与正常脑组织分界不清,手术难以彻底切除,术后极易复发。而其对放疗及化疗的敏感性均欠佳,故患者预后差,死亡率高。目前脑胶质瘤的治疗仍然是一个难题,对人类生命健康造成严重威胁,因此,寻找更好的抗胶质瘤药物具有十分重要的意义。糖尿病最易并发脑血管病变,持续的高血糖引发严重的神经毒性,使患者神经功能受损,预后不良。据统计,糖尿病合并脑缺血患者的病残率、复发率及死亡率均显著高于非糖尿病脑缺血患者,给社会和家庭带来沉重负担,已成为目前严重危害公民健康和生命的重大公共卫生问题。此外,即使无糖尿病,缺血性脑卒中后患者血糖水平也常常会升高,加重脑损伤。《中国急性缺血性脑卒中诊治指南2014))指出：“约40%的患者存在卒中后高血糖,对预后不利。”因此,除了降血糖外,迫切需要有效措施抑制高血糖毒性,改善神经病损,促进神经功能恢复,进而提高临床治疗效果。脑卒中是人类灾难性疾病,具有高发病率、高死亡率和高致残率等特点,是世界范围内死亡和长期致残的三大原因之一,同时也是世界上单病种导致成人后天功能障碍的首位原因。2008年第三次全国死因调查表明,脑卒中已跃居为我国人口死亡原因的第一位；在各种原因所致死亡中,我国死于脑卒中的构成比是欧美发达国家的4-5倍。脑缺血再灌注损伤机制复杂,氧化还原平衡的破坏是其重要的病理特征。因此,探索高效的抗氧化神经保护剂对脑缺血再灌注损伤的改善及治疗具有重要的研究意义和临床价值。硒是人体必需的微量元素,对人体生命活动必不可少,可发挥抗氧化、抗肿瘤、提高免疫力、拮抗重金属等多种作用。已证实,硒对维持正常神经功能发挥有重要作用；而硒缺乏可影响神经认知功能及导致神经退行性变,如阿尔茨海默病、帕金森病。大量临床及动物研究证据显示,高剂量的硒能加剧氧化应激,通过诱导活性氧族物质(reactive oxygen species, ROS)的累积诱导氧化损伤,发挥促凋亡的功效；而低剂量的硒则具有拮抗氧化应激、抑制细胞凋亡的作用。故人们常常采用高剂量硒作为肿瘤化疗制剂,诱导肿瘤细胞周期阻滞,甚至凋亡,以达到抑癌、抗癌目的；而采用低剂量硒作为细胞保护剂,对抗机体内的氧化应激损伤。硒代胱氨酸(selenocystine, SeC)是自然可获取的小分子有机硒化合物,被称为人类第21个必需氨基酸,因其多重药理学功效得到广泛应用,尤其是其对氧化还原通路的调节引起众多研究者的高度关注。3,3’-二硒二丙酸(3,3'-diselenodipropionic acid, DSePA)是SeC的衍生物,性质稳定、安全性高,具有高效的抗氧化活性,广泛用于抗氧化研究,在体外和体内实验中对急性神经毒性和慢性神经退行性病变均表现出保护效果。据报道,硒缺乏可大大增加机体患癌风险,而补充硒可降低某些癌症的发病率。SeC具有广谱的抗肿瘤活性,可通过诱导凋亡在体内、外抑制多种人类肿瘤细胞生长,如肝癌、肺腺癌、乳腺癌及黑色素细胞瘤,具有潜在的临床应用价值。近几年研究还发现：硒化合物联合抗肿瘤药物一起使用,可增强抗肿瘤药物的敏感性,即硒化合物可作为肿瘤化疗的增敏剂。然而,SeC是否对脑胶质瘤细胞的生长具有抑制作用,其机制如何,目前尚未见有报道。大脑属于高耗氧器官,神经元对葡萄糖的高消耗必然伴随自由基的大量产生；而高血糖会破坏细胞内的抗氧化酶系,削弱其清除能力,使大量自由基无法被及时清除。持续高血糖可影响和破坏线粒体内膜,启动线粒体膜电位耗散,导致大量自由基外泄。过多自由基的沉积,会氧化质膜,破坏神经突触,扰乱神经元之间的连接,甚至导致神经元凋亡。实验证实,拮抗高血糖诱导的氧化应激损伤可明显减弱神经损伤／毒性,改善神经病变。然而,有机硒是否对高糖诱导的神经毒性有拮抗作用,其机制如何,目前尚未见有报道。急性缺血性脑卒中发作中,脑缺血再灌注损伤的病理机制复杂,氧化还原平衡的破坏是其重要的病理特征。氧化应激通过ROS超载导致脂质过氧化和DNA损伤,在缺血再灌注脑损伤中发挥关键作用。因此,探索高效的抗氧化神经保护剂对脑缺血再灌注损伤的改善及治疗具有重要的研究意义和临床价值。而硒对脑缺血再灌注的直接保护作用,尚未见有研究报道。故本课题开展了在神经系统疾病方面,SeC及其衍生物DSePA的作用研究,探讨有机硒对脑胶质瘤、高糖神经毒性、缺血性脑卒中的作用效果及机理。首先,本研究选取了人脑胶质瘤细胞系U251、U87为研究对象,研究SeC对U87和U251细胞周期的影响,同时从影响肿瘤信号转导通路的重要信号蛋白MAPKs和AKT入手,分析其可能的作用机制,为人脑胶质瘤的治疗提供实验依据。其次,本研究选取PC12细胞作为神经细胞模型,选用DSePA对高糖诱导的毒性损伤进行干预,探讨其对高糖神经毒性的拮抗作用。最后,本研究构建了短暂性局灶性脑缺血再灌注(transient focal cerebral ischemia/reperfusion injury, tFCI/R)损伤小鼠模型,以SeC进行干预,探讨其对缺血再灌注损伤的保护作用及机制。目的1.探究SeC对U251、U87两种人脑胶质瘤细胞系的生长抑制效果,并探讨其潜在的分子机制。2.探究DSePA在PC12细胞中对高糖诱导神经毒性的拮抗效果及机制。3.探究SeC对tFCI/R损伤小鼠的保护功效及机制。方法1.取对数生长期U251和U87细胞体外培养,待细胞贴壁后随机分为正常对照组、各剂量SeC用药组。分别以5、10、20μM的SeC处理24 h或48 h。使用相差显微镜观察各组细胞形态学变化；采用四甲基偶氮唑蓝(MTT)比色法测定细胞生存率,了解SeC对U251和U87细胞生长的抑制；应用流式细胞术分析SeC对U251和U87细胞周期分布的影响；TUNEL-DAPI法观察SeC对U251细胞DNA的损伤情况；采用DCFH-DA探针和超氧阴离子试剂盒检测ROS和超氧阴离子的产生；Western blotting检测SeC对U251中细胞周期调控相关蛋白Cyclin A,细胞凋亡相关蛋白p21、p53及细胞内相关信号通路的蛋白水平(MAPKs及AKT)的影响。同时,应用SeC联合多种通路蛋白抑制剂研究其对细胞周期分布的影响。2.取对数生长期PC12细胞体外培养,待细胞贴壁后随机分为正常对照组、DSePA用药组,100 mM葡萄糖处理48 h制作高糖损伤细胞模型,加入DSePA进行干预。采用流式细胞分析技术及TUNEL-DAPI共染色观察细胞凋亡情况;检测caspase-3/-8/-9活性,结合Western blotting结果确定凋亡路径；通过JC-1探针评价线粒体膜电位,Mito-tracter与DAPI共染色检测线粒体结构改变,MitoSOX染料检测超氧阴离子；采用DCFH-DA检测细胞内ROS水平,并加入ROS清除剂进一步证实氧化损伤的作用；使用Western blotting法检测相关蛋白表达。3.手术制作tFCI/R小鼠模型,通过多普勒血流监测和神经功能缺损评分筛选手术合格的小鼠,随机分至SeC治疗组(SeC)和溶媒对照组(Vehicle),同时设假手术对照组(Sham,小鼠经历手术全程而不进行大脑中动脉阻塞)。SeC组小鼠给予术后连续3日,每日1次腹腔注射SeC溶液(2 mg/30 g),Vehicle组和Sham组小鼠给予同样方式注射等量生理盐水。于术后24、48、72 h各进行一次神经功能缺损评分及神经行为学测试；手术3天后行TTC染色测脑梗死体积,干-湿重法测脑水肿程度变化,免疫荧光染色观察AQP4表达情况,TUNEL-DAPI染色观察神经元凋亡情况,Western blotting检测活性caspase-3和活性caspase-9表达。结果1.MTT结果显示,SeC时间/剂量依赖性地抑制了U251和U87细胞的生长,镜下观察可见细胞变圆,突触减少,细胞数目明显减少,与正常细胞相比,差异具有统计学意义(P0.05)；流式细胞术结果表明SeC处理诱导了明显的U251和U87细胞S期细胞周期阻滞,这与cyclin A蛋白表达下调相一致；TUNEL-DAPI分析和ROS检测结果显示,SeC处理导致了ROS升高诱导的U251细胞DNA损伤；Western blotting结果表明,SeC处理显著上调了DNA损伤标志物p21和p53的表达,并引起pJNK、p38、pERK的表达上调和pAKT的表达下降。2. DSePA的预处理可有效减弱高糖对PC12细胞的毒性作用。流式细胞技术检测到,DSePA预处理可显著抑制高糖诱导的PC12细胞凋亡,表现为sub-G1峰的降低；荧光染色技术结果表明,DSePA预处理可有效抑制高糖诱导的染色质的断裂和核浓缩。caspases活性检测显示DSePA抑制了高糖诱导的caspases活性,Western blotting术从蛋白水平检测发现,高糖诱导的PARP切割和caspase-3/-7/-9激活被DSePA预处理显著抑制；线粒体膜电位及结构检测证实,DSePA阻断了高糖诱导的线粒体损伤。DSePA抑制了PC12细胞中高糖诱导的超氧阴离子产生和ROS累积。3. tFCI/R术后24、48、72 h神经功能缺损评分与神经行为学测试表明,随时间延长,所有小鼠神经功能损伤及感觉运动功能障碍均得到一定程度的改善,而SeC治疗组改善显著,与Vehicle组相比有统计学差异；SeC治疗组在手术3天后脑梗死体积和水肿程度上较Vehicle组均有明显降低,AQP4的表达比Vehicle组显著降低,表明SeC明显缓解了脑组织的梗死和水肿；TUNEL-DAPI染色、Western blotting结果表明,SeC处理显著下调了caspase-3和PARP的裂解,从而抑制了神经元凋亡。结论1.SeC剂量/时间依赖地抑制人脑胶质瘤细胞的增殖,其分子机制为通过调节MAPKs和AKT信号通路诱导人脑胶质瘤细胞S期阻滞。2.DSePA可通过抑制氧化应激诱发的细胞凋亡,拮抗高糖神经毒性,有望成为拮抗高糖诱导神经系统疾病的高效策略。3.腹腔注射SeC对小鼠脑缺血再灌注损伤确有保护功效,主要是通过抑制水肿,抵抗氧化作用诱导的神经元凋亡。
[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 .

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.41
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本文编号：1822290