Salubrinal减轻大鼠脑死亡状态下肝损伤的分子机制研究

发布时间：2018-05-29 16:34

本文选题：内质网应激 + 凋亡　；参考：《郑州大学》2015年博士论文

【摘要】：肝脏移植是治疗终末期肝脏疾病最有效的治疗途径。制约肝脏移植发展的最主要因素是肝脏供体的短缺。脑死亡供体是肝脏移植的主要供体来源。脑死亡是指包括脑干功能在内的全脑功能不可逆和永久的丧失。脑死亡是一个复杂的病理生理过程,是一个直接影响器官形态和功能的动态过程。研究表明脑死亡来源的供体较活体来源的供体移植术后并发症多、生存率低。因此,脑死亡被认为是造成供体器官损伤的一个独立危险因素。细胞凋亡的增加是导致脑死亡供体肝脏损伤的主要因素。引起细胞凋亡的途径有三条,内质网应激途径引起凋亡是线粒体途径和外源性途径之外的第三条凋亡途径。通常情况下,内质网通过未折叠蛋白反应保护由外界刺激对细胞的损伤,但是当应激时间延长或者应激强度增大,不能通过未折叠蛋白反应保护机制代偿,最终会通过激活内质网应激凋亡通路引起细胞凋亡。内质网类似激酶(PKR-like endoplasmic reticulum kinase,PERK)是一种丝氨酸苏氨酸激酶,在内质网应激启动后,通过磷酸化真核生物的翻译起始因子2α(eukaryotic translation initiation factor 2α,e IF2α),介导细胞的凋亡。Salubrinal可通过选择性诱导e IF2α磷酸化和抑制其去磷酸化来对抗内质网应激(endoplasmic reticulum stress,ERs)诱导的细胞凋亡,并且其不保护与ERs无关的细胞凋亡刺激。研究表明Salubrinal能减轻细胞的凋亡。因此,研究应用内质网应激诱导凋亡抑制剂对脑死亡肝脏损伤的影响,有利于阐明脑死亡对肝脏损伤的机制,为脑死亡下器官功能的保护奠定理论基础和提供药物靶点。目的:探讨内质网应激抑制剂Salubrinal对大鼠脑死亡状态下肝损伤的影响及分子机制方法:本研究分为三个部分:第一部分:大鼠脑死亡模型制作方法的改进:在传统渐进式诱导脑死亡模型的基础上进行改良,采用缓慢间歇颅内加压法,诱导脑死亡的发生。通过以下标准进行脑死亡的判定:深昏迷、脑干反射消失、自主呼吸停止及脑电图出现平直。在诱导脑死亡发生及维持脑死亡状态时实时监测颅内压、脑电图、心电图、平均动脉压、体温等的变化。第二部分:内质网应激介导脑死亡状态下肝损伤的机制研究:在改良的大鼠脑死亡模型的基础上,将大鼠随机分为假手术组及脑死亡组。通过免疫组织化学及蛋白质免疫印迹检测假手术组及脑死亡组大鼠肝脏中内质网应激分子标志物(Grp78和Xbp-1)及凋亡相关蛋白(Chop和Caspase-12)的变化。另外,利用原位细胞凋亡法检测肝细胞的凋亡及透射电镜观察肝细胞的微观变化。通过荧光定量PCR检测内质网应激凋亡相关基因的m RNA表达变化。第三部分:Salubrinal对大鼠脑死亡状态下肝损伤的作用:将SD大鼠随机分为3组:脑死亡组;脑死亡加DMSO组;脑死亡加Salubrinal组。脑死亡加DMSO组在诱导脑死亡前1小时腹腔注射DMSO;脑死亡加Salubrinal组在诱导脑死亡前1小时腹腔注射Salubrinal。q PCR检测大鼠肝脏中Chop及Caspase-12 m RNA的表达;Western blot检测大鼠肝脏中PERK、p-e IF2α、e IF2α、Chop及Caspase-12蛋白的表达;免疫组化检测Chop及Caspase-12在大鼠肝组织中分布和表达;全自动生化分析仪检测ALT及AST的表达;TUNEL法检测肝细胞的凋亡。结果:第一:建立了稳定的大鼠脑死亡模型:经有效的呼吸循环支持均能成功诱导脑死亡的发生并且均能维持脑死亡状态6小时以上,平均动脉压在80mm Hg以上;第二:内质网应激可能介导大鼠脑死亡状态下的肝细胞损伤:脑死亡可以导致内质网应激的标志物Grp78和Xbp-1表达增加,以及内质网应激相关的凋亡蛋白Chop和Caspase-12表达增加。另外,通过透射电镜发现肝细胞出现细胞凋亡的特征以及通过原位细胞凋亡法检测发现肝细胞凋亡增加;第三:Salubrinal减轻大鼠脑死亡状态下的肝损伤:脑死亡组及DMSO加脑死亡组在Chop及Caspase-12的m RNA及蛋白表达水平以及PERK、e IF2α及p-e IF2α的蛋白表达没有统计学差异。Salubrinal干预后相对脑死亡组p-e IF2α表达升高,e IF2α表达没有明显改变而P-PERK在脑死亡2h及6h表达降低。q PCR结果显示:Salubrinal干预后Chop的m RNA表达在脑死亡4h明显降低,Caspase-12的m RNA表达也明显降低。我们也通过Western blot及免疫组织化学检测发现:Salubrinal干预后Chop及Caspase-12的蛋白表达也明显下降。此外,Salubrinal干预后,肝功得到改善,肝细胞的凋亡减轻。结论:经改良的缓慢间歇颅内加压法建立的大鼠脑死亡模型,较传统的脑死亡模型相比具有维持脑死亡状态时间长、简单易复制、易于标准化、与临床脑死亡相似的特点。首次发现大鼠脑死亡状态下启动内质网应激,并且内质网应激的凋亡途径介导脑死亡下的肝损伤。首次发现Salubrinal能明显减轻脑死亡大鼠肝细胞的凋亡,可能通过PERK/e IF2α通路来实现其保护作用。
[Abstract]:Liver transplantation is the most effective treatment for end-stage liver disease. The main factor restricting the development of liver transplantation is the shortage of liver donor. Brain death donor is the main source of liver transplantation. Brain death refers to the irreversible and permanent loss of whole brain function including brain stem function. Brain death is a complex disease. Physical and physiological processes are a dynamic process that directly affects organ morphology and function. The study shows that brain death donors have more complications and lower survival rates than donor donor transplantation. Therefore, brain death is considered as an independent risk factor for donor organ damage. The increase of cell apoptosis is the leading brain death donor. The main factors of liver injury. There are three ways to induce apoptosis. The endoplasmic reticulum stress pathway causes apoptosis as the third apoptotic pathway outside the mitochondrial pathway and exogenous pathway. In general, the endoplasmic reticulum protects the cells by external stimulation by the reaction of unfolded proteins, but when the stress time is prolonged or Ying Jiqiang is prolonged. PKR-like endoplasmic reticulum kinase (PERK) is a serine threonine kinase (serine reticulum kinase, PERK). After the endoplasmic reticulum should be stimulated, the translational initiation of eukaryotic phosphorylated eukaryotes is initiated. Factor 2 alpha (eukaryotic translation initiation factor 2 alpha, e IF2 a), mediated cell apoptosis.Salubrinal can be induced by selectively inducing e IF2 alpha phosphorylation and inhibiting its dephosphorylation to counter endoplasmic reticulum stress induced apoptosis, and it does not protect cell apoptosis that is irrelevant. It shows that Salubrinal can reduce cell apoptosis. Therefore, the study of the effect of endoplasmic reticulum stress induced apoptosis inhibitor on brain death in the liver injury is beneficial to elucidate the mechanism of brain death to liver injury, lay a theoretical foundation for the protection of organ function under brain death and provide drug targets. Objective: to explore the endoplasmic reticulum stress inhibitor Salubri The effect and molecular mechanism of nal on brain death in rats: This study is divided into three parts: the first part: the improvement of the method of making brain death model in rats: Based on the traditional progressive induced brain death model, a slow intermittent intracranial pressure method is used to induce the occurrence of brain death. Determination of brain death: deep coma, brain stem reflex disappearance, autonomic breathing stop and electroencephalogram straight. The changes of intracranial pressure, electroencephalogram, electrocardiogram, mean arterial pressure and body temperature are monitored in real time when inducing brain death and maintenance of brain death. The second part: the mechanism of endoplasmic reticulum stress mediated liver injury in brain death: On the basis of the modified rat brain death model, rats were randomly divided into sham operation group and brain death group. The changes of endoplasmic reticulum stress molecular markers (Grp78 and Xbp-1) and apoptosis phase Guan Danbai (Chop and Caspase-12) in sham operation group and brain death group were detected by immunohistochemistry and protein immunoblotting. In situ apoptosis assay was used to detect the apoptosis of hepatocytes and the microscopic changes of liver cells by transmission electron microscopy. The changes in M RNA expression of endoplasmic reticulum stress apoptosis related genes were detected by fluorescence quantitative PCR. The third part: the effect of Salubrinal on liver injury in the brain death of rats: the SD rats were randomly divided into 3 groups: brain death group; brain death plus DMSO Group: brain death plus Salubrinal. Brain death and group DMSO were intraperitoneally injected with DMSO 1 hours before inducing brain death; brain death and Salubrinal group were intraperitoneally injected with Salubrinal.q PCR to detect the expression of Chop and Caspase-12 m RNA in rats' liver 1 hours before the induction of brain death; Western blot was used to detect the liver in rat liver. The expression of -12 protein; immunohistochemical detection of Chop and Caspase-12 in rat liver tissue distribution and expression; full automatic biochemical analyzer to detect the expression of ALT and AST; TUNEL method to detect the apoptosis of liver cells. Results: First: a stable rat brain death model was established: the brain death could be successfully induced by effective respiratory cycle support. All of them can maintain brain death for more than 6 hours and average arterial pressure above 80mm Hg; second: endoplasmic reticulum stress may mediate hepatocyte injury in rat brain death: brain death can lead to the increase of Grp78 and Xbp-1 expression of endoplasmic reticulum stress, and the increase of apoptosis protein Chop and Caspase-12 expression related to endoplasmic reticulum stress. In addition, the apoptosis of hepatocytes was found by transmission electron microscopy and apoptosis was detected by in situ cell apoptosis. Third: Salubrinal alleviated liver injury in the brain death of rats: the m RNA and protein expression levels of the brain death group and the DMSO brain death group in Chop and Caspase-12, and PERK, e IF2 alpha and P-E. There was no statistical difference in the expression of IF2 alpha protein. The expression of P-E IF2 alpha in the relative brain death group increased after.Salubrinal intervention, and the expression of E IF2 alpha was not significantly changed, while the P-PERK in 2H and 6h expression in brain died and.Q PCR showed that the expression of E was significantly lower in brain death. We also found that the protein expression of Chop and Caspase-12 was also significantly decreased after the Salubrinal intervention by Western blot and immunohistochemistry. Furthermore, the liver function was improved and the apoptosis of hepatocytes was alleviated by the prognosis of Salubrinal. Conclusion: the rat brain death model established by the modified slow intermittent intracranial pressure method was compared with the traditional brain death model. It has a long time of maintaining brain death, simple replicating, easy to standardize and similar to clinical brain death. It is the first time that endoplasmic reticulum stress is initiated in rat brain death, and the apoptosis pathway of endoplasmic reticulum stress mediates the liver damage under brain death. The first occurrence of Salubrinal can significantly reduce the liver cells of brain death rats. Apoptosis may be protected by PERK/e IF2 alpha pathway.
【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】：R657.3

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