内质网应激参与脑死亡状态下肝损伤的分子机制研究

发布时间：2018-05-31 11:10

本文选题：颅脑损伤 + 脑死亡　；参考：《郑州大学》2015年博士论文

【摘要】：背景与目的颅脑损伤(head injury)是临床上导致脑死亡(brain death,BD)的主要原因之一。脑死亡是指包括脑干在内的全脑功能丧失的不可逆转的状态。脑死亡概念是1959年由法国学者P.Mollaret和M.Goulon在第23届国际神经学会上首次提出并使用,用来描述这种严重大脑损伤后失去反射需要机械呼吸支持的不可逆昏迷的状态。1966年美国学者提出脑死亡是临床死亡的标志。在1968年在第22届世界医学大会上,美国哈佛医学院脑死亡定义审查特别委员会提出“脑功能不可逆性丧失”作为新的死亡标准,并制定了世界上第一个脑死亡诊断标准,法律上承认脑死亡等同于死亡。目前,世界大多数发达国家已认可脑死亡,并相继颁布脑死亡法,从法律上保障脑死亡临床诊断。我国2003年中华医学杂志等主要医学杂志刊登了卫生部脑死亡判定标准起草小组制订的《脑死亡判定标准(成人)(征求意见稿)》和《脑死亡判定技术规范(成人)(征求意见稿)》。2009年4月,经过修改与完善,其修订稿发表,比较详细,具有可操作性。由于脑死亡是一个涉及医学、伦理学、法学、社会学等多个学科的概念,尽管呼声很高,我国实现脑死亡立法还有许多实际问题,但将是一种必然趋势。脑死亡供体是联合国推荐优先发展的器官移植供体,动物模型和临床研究均已证明脑死亡是一个直接影响器官形态和功能的动态过程,其机制仍然不能完全明了。研究表明,脑死亡可引起心、肾细胞凋亡增加。进而引起形态改变和功能受损。2003年发表于《Transplantation》杂志上的关于脑死亡的文章,进一步揭示脑死亡诱导肝细胞凋亡增加,且以肝实质细胞为主,其机制与死亡受体途径和线粒体途径均有关。另有多项研究证实,凋亡是造成脑死亡供体器官移植后生存力下降的原因之一。最近的研究表明,内质网应激(endoplasmic reticulum stress,ERs)也是细胞凋亡调节中的重要环节之一。内质网(endoplasmic reticulum,ER)在细胞内分布广泛,参与重要生理功能的维持,主要负责蛋白质的合成转运、信号肽识别、糖基化修饰等过程和钙离子的贮存,信号转导及细胞内钙的再分布。内质网巨大的膜结构在细胞内提供了一个宽广的分子组装、反应平台,使之在多种信号调控中起到关键作用。内质网的功能对大多细胞的活动和生存是必需的。ERs途径是独立于细胞膜受体或线粒体途径的第3条凋亡信号途径。ER对诸如能干扰ATP、氧化状态及Ca2+浓度等的内环境改变非常敏感,这些改变会降低内质网蛋白折叠能力,引起未折叠蛋白的积累和聚集,从而激活未折叠蛋白反应(unfolded protein response,UPR),以保护由ERs所引起的细胞损伤,恢复细胞功能。但当损伤超过修复能力时,ERs则引起细胞凋亡。以ERs相关生物大分子作为靶点进行药物研究将为相关疾病的预防和治疗开辟新的途径。许多肝脏疾病的发病机制均与ERs引起的损伤有关,但ERs是否参与、介导脑死亡状态下肝损伤、肝细胞凋亡的过程至今未见文献报道。如能证实ERs参与了该过程的介导,并找到针对ERs途径的干预措施来减轻肝损伤,将为脑死亡后器官功能保护提供新的思路,从而为临床肝移植提供潜在的供体。本课题拟分三个部分从组织及细胞水平揭示内质网应激参与SD大鼠脑死亡状态下肝损伤的分子机制。第一部分:SD大鼠脑死亡模型的建立与维持目的本研究拟在我们前期研究的基础上进一步探索采用缓慢间歇颅内加压法模拟颅内出血、脑疝形成建立稳定的Sprague Dawley(SD)大鼠脑死亡模型以及较长时间维持脑死亡状态的方法,为研究SD大鼠脑死亡状态下的肝脏形态和功能变化及其他相关基础、临床研究提供稳定的实验动物模型。材料与方法实验动物:成年,雄性,健康,SD大鼠,体重250g~350g,自由饮食。实验方法:颅骨钻孔,硬脑膜外腔置入Fogarty 3F气囊导管,其尖端指向大鼠尾侧,气囊导管连接球囊压力注射泵,通过向气囊注入生理盐水进行颅内加压,直至达到脑死亡状态。脑死亡判断标准:(1)深昏迷;(2)角膜反射消失;(3)瞳孔固定、散大;(4)静息脑电图;(5)自主呼吸消失。观察指标:全程记录生命体征(心率HR、呼吸R、体温T、血压BP)、尿量、血流动力学、脑电图、脉搏氧饱和度(SPO2)及其与颅内压变化的相关规律。实验分组:健康SD大鼠随机分为:①对照组(C组):与脑死亡组对应,每个时间点n=10只,腹腔注射麻醉后隐动脉置管(24G静脉留置针,行有创动脉血压监测)、气管插管、颅骨钻孔并置入Fogarty 3F导管,并实时监测动脉压、脉搏氧饱和度、体温、脑电图变化,颅内不加压不建立脑死亡模型;②脑死亡组(BD组),按实验设计分为脑死亡0h、1h、2h、4h、6h时间点组,每时间点组10只,除以上操作外,应用缓慢间歇颅内加压法建立脑死亡模型,通过呼吸、循环支持维持实验动物脑死亡状态0h、1h、2h、4h、6h,维持脑死亡状态要求大鼠生命体征稳定:实时监测平均动脉血压(mean artery pressure,MAP)≥80mm Hg,体温38.1±0.2℃,脉搏氧饱和度≥95%,呼吸机机械通气支持,适当增加吸入气中氧含量,呼吸频率、潮气量由HARVARD动物呼吸机自动据体重计算参数,可适当微调整,脑电图呈静息状态,血流动力学稳定。结果 1.大鼠脑死亡模型的稳定建立采用缓慢间歇颅内加压法可模拟脑出血病理生理过程建立简便、易重复的SD大鼠脑死亡模型,按实验要求稳定保持脑死亡状态6h,为后续相关研究提供可靠的动物模型。模型建立过程详见图1-2,3,4,5,6,7,8。2.平均动脉压(MAP)的变化脑死亡各组显示相似的平均动脉压变化过程,并与文献报道结果一致。脑死亡判定后,在呼吸机支持的情况下,适当调整吸入气中氧浓度,脑死亡模型血流动力学稳定,平均动脉压维持在80mm Hg以上,实验全程不使用血管活性药物。与对照组相比较,差异有统计学意义(P0.05)。(图1-14)3.心率的变化脑死亡过程心率出现典型的变化,每个实验组内动物模型出现相同的变化过程,与对照组相比较,差异具有统计学意义(P0.05)。详见图1-13。4.脑电图的变化随着缓慢颅内加压诱导开始,脑电活动开始紊乱,脑电压不稳定,随着颅内压的增高,脑电活动逐渐减弱,渐成一直线。脑死亡组各时间点组均出现相似的脑电变化过程,与对照组比较,差异显著。详见图1-10,11,12。第二部分:探讨脑死亡状态下SD大鼠肝脏形态与功能的变化目的观察脑死亡状态下SD大鼠肝脏形态与功能的变化特点,研究脑死亡对肝脏的影响。方法脑死亡组SD大鼠在脑死亡判定后0h、1h、2h、4h、6h分别停止呼吸支持,剖腹,于下腔静脉处抽静脉血,室温静止、离心后取血清分装冻存管后-20℃或-80℃冻存待测;取相同部位肝组织分别:①迅速置入冻存管液氮保存;②切成小颗粒状(0.5-1mm3)置入4%戊二醛液固定,4℃冷藏备电镜切片;③肝组织切成片状(约1-2cm2大小,厚度为0.2-0.3cm)放入中性福尔马林溶液浸泡制成石蜡块。应用全自动生化分析仪检测血清肝脏酶学(ALT、AST)的变化;透射电镜(TEM)观察肝脏形态学变化;罗氏公司原位末端凋亡检测试剂盒观察肝组织凋亡细胞。对照组SD大鼠按上述时间点同样方法取材。结果 1.脑死亡组大鼠0h、1h、2h、4h、6h不同时间点血清ALT、AST变化脑死亡诱导判定后SD大鼠肝功能(ALT、AST)出现不同程度的变化,随时间的推移,ALT、AST呈逐渐升高趋势,与对照组相比较,差异具有统计学意义(附表2-1)。2.电镜下观察肝细胞形态结构变化对照组SD大鼠肝组织细胞未见明显异常,脑死亡组SD大鼠随时间推移,肝组织结构出现损伤性变化,且逐渐加重,如轻度损伤为线粒体轻度肿胀,线粒体脊欠清晰,线粒体内外膜破损(BD-2h),中度损伤表现为部分线粒体脊损伤(BD-4h),重度为线粒体脊融合,内外膜破损,线粒体肿胀明显,线粒体增生且膜性结构融合(附图2-1)。3.肝组织肝细胞凋亡检测对照组大鼠肝脏TUNEL染色少见凋亡。脑死亡组,SD大鼠肝脏细胞凋亡数量明显增加,且随脑死亡维持时间的延长,肝细胞凋亡细胞逐渐增加(附图2-2,3)。第三部分:SD大鼠脑死亡过程是否启动肝细胞内质网应激;内质网应激是否介导或参与脑死亡状态下肝损伤、肝细胞凋亡过程目的探讨SD大鼠脑死亡过程是否启动肝细胞内质网应激,研究内质网应激是否介导或参与脑死亡状态下肝损伤、肝细胞凋亡过程。方法分别采用Western blot及实时荧光定量PCR(real-time quantitative polymerase chain reaction,q PCR)、免疫组化(immunohistochemistry,IHC),对各实验组及不同时间点组肝组织标本进行GRP78;XBP-1;CHOP;JNK;Caspase-12;ATF4基因及其部分蛋白表达分析。结果与对照组相比,脑死亡状态下SD大鼠肝组织不同时间点随时间推移内质网应激相关大分子物质GRP78/Bi P;XBP-1;CHOP/GADD153;JNK;Caspase-12;ATF4从基因水平均表达增加,其中GRP78、CHOP、XBP-1、Caspase-12蛋白表达明显增加,初步表明脑死亡过程诱导SD肝组织损伤,并启动内质网应激、未折叠蛋白反应,参与肝细胞损伤过程,差异具有显著性,具有统计学意义。(附图2-4,5,6,7,8,9,10,11,12,13,14)结论 1.采用缓慢间歇颅内加压法可建立稳定的SD大鼠脑死亡模型,为进行下一步动物实验提供可靠的基础。2.脑死亡过程对SD大鼠肝脏有损伤作用,引起肝细胞凋亡。3.脑死亡过程启动SD大鼠肝细胞内质网应激,参与肝细胞损伤、肝细胞凋亡过程。
[Abstract]:Background and objective craniocerebral injury (head injury) is one of the main causes of the clinical brain death (BD). Brain death is an irreversible state of the loss of whole brain function including the brain stem. The concept of brain death was first proposed and used by French scholars P.Mollaret and M.Goulon at the twenty-third International Neuroscience Society in 1959. To describe the state of irreversible coma that lost reflection and need mechanical respiratory support after the severe brain damage, American scholars have suggested that brain death is a sign of clinical death in.1966. At the twenty-second world medical conference in 1968, the brain death definition Review Committee of the Harvard Medical School proposed "the loss of brain function irreversible loss." "As the new standard of death, the first diagnostic standard of brain death in the world has been established, and it is legally recognized that brain death is equivalent to death. At present, most of the developed countries in the world have recognized brain death and promulgated the brain death method successively to ensure the clinical diagnosis of brain death. The major medical journals such as the Chinese Medical Journal of China in 2003 published the major medical journals published in China. The "brain death criteria (adult)" (Draft) and the technical specification for brain death (solicitation draft), formulated by the drafting group of the Ministry of health's brain death criteria (adult), and the technical specification for brain death (solicitation draft), in April >.2009, were revised and perfected, and their revised manuscripts are more detailed and operable. Brain death is a medical, ethical, and jurisprudential process. There are many practical problems in the realization of brain death legislation in China, although the concept of sociology and other disciplines has many practical problems, but it will be an inevitable trend. The brain death donor is the priority of the organ transplant donor recommended by the United Nations. Animal models and clinical studies have proved that brain death is a direct influence on the dynamics of organ morphology and function. The study shows that the mechanism of the brain death can not be completely clear. The study shows that brain death can cause the heart, the apoptosis of the kidney cells increase. Then the morphological changes and function damage are caused by.2003 published in the Journal of

on brain death, which further reveals that brain death induces the increase of apoptosis in the liver cells, and the mechanism of the liver parenchyma cells is the main mechanism. A number of studies have confirmed that apoptosis is one of the reasons for the decrease of viability after brain death donor organ transplantation. Recent studies have shown that endoplasmic reticulum stress (ERs) is also one of the important links in the regulation of apoptosis. The endoplasmic reticulum (endoplasmic reticul) Um (ER) is widely distributed in cells and participates in the maintenance of important physiological functions. It is responsible for the synthesis and transport of protein, signal peptide recognition, glycosylated modification, storage of calcium ions, signal transduction and the redistribution of intracellular calcium. The large membrane structure of the endoplasmic reticulum provides a broad molecular assembly, reaction platform, The function of the endoplasmic reticulum is essential to the activity and survival of most cells. The.ERs pathway is the third apoptotic signaling pathway independent of the cell membrane receptor or mitochondrial pathway,.ER, which is very sensitive to internal environmental changes such as ATP, oxidative state and Ca2+ concentration. These changes will decrease. The folding ability of the meshwork proteins causes the accumulation and aggregation of unfolded proteins to activate the unfolded protein reaction (unfolded protein response, UPR) to protect cell damage caused by ERs and restore cell function. But when the damage exceeds the repair capacity, ERs causes apoptosis. The drug is targeted by the ERs related macromolecules. The research will open a new way for the prevention and treatment of related diseases. The pathogenesis of many liver diseases is related to the damage caused by ERs, but whether ERs is involved, mediating the liver injury under brain death, and the process of hepatocyte apoptosis have not been reported. For example, it can be proved that ERs is involved in the process, and the ERs pathway is found. Intervention measures to alleviate liver injury will provide new ideas for the protection of organ function after brain death and provide a potential donor for clinical liver transplantation. This topic is to be divided into three parts to reveal the sub mechanism of endoplasmic reticulum stress participation in the brain death of SD rats from the tissue and cell levels. The first part: the brain death model of SD rats On the basis of our previous study, the purpose of this study is to further explore the use of slow intermittent intracranial pressure to simulate intracranial hemorrhage, brain hernia formation to establish a stable Sprague Dawley (SD) rat brain death model and a longer time to maintain the brain death state, in order to study the liver shape in the brain death of SD rats. State and function changes and other related bases, clinical studies provide a stable experimental animal model. Materials and methods experimental animals: adult, male, healthy, SD rats, body weight 250g~350g, free diet. Experimental methods: cranial drilling, Fogarty 3F balloon catheter in the extradural cavity, its tip pointing to the tail of rat, balloon catheter connected balloon pressure Force injection pump, intracranial pressure was injected into the air bag until cerebral death was achieved. Brain death criteria: (1) deep coma; (2) disappearance of corneal reflex; (3) pupil fixed, loose; (4) resting electroencephalogram; (5) spontaneous breathing disappearance. The whole record of life signs (heart rate HR, respiratory R, temperature T, blood pressure BP), urine volume, blood Flow mechanics, electroencephalogram, pulse oxygen saturation (SPO2) and the changes in intracranial pressure. Experimental groups: healthy SD rats were randomly divided into: 1. Control group (group C): corresponding to brain death group, n=10 only at each time point, intraperitoneal injection of saphenous artery after anesthesia (24G venous indwelling needle, invasive arterial blood pressure monitoring), tracheal intubation, cranium drill The Fogarty 3F catheter was inserted into the hole, and the arterial pressure, pulse oxygen saturation, temperature, electroencephalogram and brain death model were not established, and the brain death group (group BD) was divided into group of brain death 0h, 1H, 2h, 4h, 6h time point group with 10 rats at each time point, in addition to the above operation, the brain was established by the slow intermittent intracranial pressure method. Death model, through breathing, and circulation support to maintain the brain death state of experimental animals 0h, 1H, 2h, 4h, 6h, to maintain the brain death state of the rat's vital signs is stable: real-time monitoring of the average arterial blood pressure (mean artery pressure, MAP) > 80mm Hg, temperature 38.1 + 0.2 C, pulse oxygen saturation more than 95%, ventilator mechanical ventilation support, appropriate increase of inhalation gas The content of oxygen, the frequency of respiration, and the volume of tidal gas were adjusted by the HARVARD animal ventilator automatically according to the weight calculation parameters. The electroencephalogram was resting state and the hemodynamics was stable. Results the stability of the brain death model in the 1. rats was established by using the slow intermittent intracranial pressure method to simulate the pathophysiological process of cerebral hemorrhage, which was easy to repeat and the SD was easy to repeat. The rat brain death model was maintained to keep the brain death state 6h steadily and provide a reliable animal model for subsequent related studies. The process of model establishment was detailed to see the change of mean arterial pressure in each group of brain deaths of figure 1-2,3,4,5,6,7,8.2. (MAP). Under the condition of ventilator support, the oxygen concentration in the inhaled gas was properly adjusted, the hemodynamics of the brain death model was stable, the mean arterial pressure was maintained above 80mm Hg, and the vasoactive drugs were not used in the whole course of the experiment. The difference was statistically significant (P0.05). (Figure 1-14) 3. heart rate changes were typical changes in the heart rate of the brain death process. The animal models in each experimental group had the same change process, and compared with the control group, the difference was statistically significant (P0.05). The change of electroencephalogram (EEG) in figure 1-13.4. began with the slow induction of intracranial pressure, and the EEG activity began to be disturbed and the brain voltage was unstable. With the increase of intracranial pressure, the EEG activity gradually weakened and gradually became a straight line. The brain death group had similar electroencephalogram changes in each time point group. Compared with the control group, the difference was significant. See figure 1-10,11,12. second part: To explore the changes of liver morphology and function in SD rats under brain death. Objective To observe the changes of liver morphology and power in SD rats under brain death and the effect of brain death on the liver Methods SD rats in the brain death group were treated with 0h, 1H, 2h, 4h, and 6h respectively after the death of the brain, caesarean section, venous blood from the inferior vena cava, at room temperature at room temperature. After centrifugation, the serum was collected at -20 C or -80 C after the cryopreservation, and the same part of the liver tissue was immediately placed in the cryopreservation tube and stored in the liquid nitrogen; and second cut into small granular (0.5-1mm) 3) implantation of 4% glutaraldehyde solution and frozen section at 4 C; 3. Liver tissue cut into flakes (about 1-2cm2 size, thickness 0.2-0.3cm) and immersed in neutral formalin solution to make paraffin blocks. The changes of serum liver enzyme (ALT, AST) were detected by automatic biochemical analyzer; transmission electron microscopy (TEM) was used to observe the changes of liver morphology; Roche company The apoptotic cells in liver tissue were observed by the in situ terminal apoptosis detection kit. The same method was used in the control group of SD rats at the same time point. Results the serum ALT of 0h, 1H, 2h, 4h, 6h at different time points in the 1. brain dead rats was observed at different time points, and the liver function (ALT, AST) of SD rats was changed in varying degrees after the AST changes were induced by the brain death induction. Gradually increasing trend, compared with the control group, the difference was statistically significant (Appendix 2-1). The morphological changes of hepatocytes in the control group were observed under.2. electron microscope. The liver tissue cells in the control group of SD rats were not obviously abnormal. The SD rats in the brain death group changed with time, and the liver tissue structure appeared to be damaged and gradually aggravated, such as mild injury to mitochondria. Swelling, unclear mitochondrial spinal cord, mitochondrial internal and external membrane damage (BD-2h), moderate damage to mitochondrial spinal cord injury (BD-4h), severe mitochondrial spinal fusion, internal and external membrane breakage, mitochondria swelling, mitochondrial proliferation and membrane structure fusion (appended 2-1).3. liver cell apoptosis detection in the control group of rat liver TUNEL staining is rare. Apoptosis in the brain death group, the number of apoptosis in the liver cells of SD rats increased significantly, and with the prolongation of the time of the death of the brain, the number of apoptotic cells in the liver gradually increased (appended 2-2,3). The third part: whether the brain death process of SD rats started the endoplasmic reticulum stress in the liver cells; whether endoplasmic reticulum stress mediates or participates in the liver injury in the state of brain death, and the liver cell withered. The purpose of the death process is to investigate whether the brain death process of SD rats initiate the endoplasmic reticulum stress and whether endoplasmic reticulum stress mediates or participates in the liver injury and the process of hepatocyte apoptosis in the state of brain death. Methods of Western blot and real-time quantitative PCR (real-time quantitative polymerase chain reaction, Q PCR), immunohistochemistry (I) Mmunohistochemistry, IHC), GRP78, XBP-1; CHOP; JNK; Caspase-12; ATF4 gene and some protein expression analysis were carried out in the experimental group and the different time point group. The results were compared with the control group. The results were compared with the control group, and the liver tissue of SD rats at different time points was at any time in the stage of the endoplasmic reticulum stress related macromolecules GRP78/Bi P; XBP-1; XBP-1; The expression of HOP/GADD153, JNK, Caspase-12 and ATF4 increased from the gene level, in which the expression of GRP78, CHOP, XBP-1, Caspase-12 protein increased obviously. It showed that the brain death process induced the injury of the SD liver tissue, and started the endoplasmic reticulum stress, unfolded protein reaction, and participated in the liver cell damage process. The difference was significant and was statistically significant. (Figure 2-). 4,5,6,7,8,9,10,11,12,13,14) conclusion 1. a stable SD rat model of brain death can be established by the slow intermittent intracranial pressure method, which provides a reliable basis for the next animal experiment to provide a reliable basis for the damage of the.2. brain death process to the liver of SD rats, and cause the apoptosis of the hepatocyte apoptosis.3. brain to initiate the endoplasmic reticulum stress of the SD rat liver cells and participate in the liver cell apoptosis. Hepatocyte injury and the process of hepatocyte apoptosis.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R651.15

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