褪黑素对肝硬化小鼠肝脏缺血再灌注损伤的保护作用研究

发布时间：2018-05-25 10:04

  本文选题：褪黑素 + 肝硬化　； 参考：《第二军医大学》2014年硕士论文

【摘要】：目的： 本研究目的在于研究褪黑素对肝硬化小鼠肝脏缺血再灌注损伤的保护作用,研究褪黑素对肝硬化缺血再灌注损伤后生存率的影响、对肝功能的影响、对细胞凋亡率的影响、对氧化应激的影响、以及对炎症因子表达的影响；分析炎症因子表达同缺血再灌注损伤的关系,探讨褪黑素作为缺血再灌注损伤保护剂的可能。 背景： 研究显示肝癌患者中半数以上合并有肝硬化,肝功能储备下降。与正常肝脏相比,肝硬化背景下的肝脏对术中缺血再灌注损伤更加不耐受,围手术期肝功能衰竭发生率及患者死亡率高于正常肝脏人群。缺血再灌注造成肝脏伤害的主要原因包括再灌注过程中三磷酸腺苷代谢产物产生的大量活性氧分子对肝组织造成的直接损伤,以及活性氧分子激活炎症通路,导致大量炎症因子释放,导致肝细胞凋亡坏死。如果能够在缺血再灌注之前预防性的使用氧自由基清除剂,清除活性氧分子则可以减轻缺血再灌注对肝组织造成的损伤。褪黑素是有松果体及肝脏等多个器官都可以分泌的小分子物质,具有调节机体节律性生理活动的作用,以往的研究强调褪黑素对中枢神经系统的作用,近年来褪黑素的抗氧化能力得到重视。褪黑素具有强大的抗氧化能力,具有分子量小,脂溶性高,分布广泛,广谱抗氧化,毒性小安全性高等优点,能够清除氧自由基,减轻正常肝脏缺血再灌注损伤。但是肝硬化肝脏中本身存在一定的炎症分子表达,血液分布同正常肝脏也有不同,细胞代谢水平同正常肝脏也有区别,目前对肝硬化肝脏缺血再灌注损伤的研究较少。本研究目的在于探讨证实褪黑素预处理是否具有减轻肝硬化肝脏缺血再灌注损伤作用,并进一步探讨其具体机制。 方法： 100只BALB/C小鼠随机分为四氯化碳(CCL4)灌胃诱导肝硬化组和正常肝对照组,肝硬化组又分为肝硬化缺血再灌注褪黑素预处理组和肝硬化缺血再灌注对照组。生存率检测分三大组,分别是空白组,肝硬化组和肝硬化褪黑素预处理组。外科手术过程为4%水合氯醛麻醉后,逐层开腹,两根无菌脱脂棉签暴露肝门,动脉夹选择性夹闭门静脉左支即选择性阻断小鼠肝中叶及肝左叶入肝血流,约5秒钟后肝中叶及左叶颜色迅速变暗,提示阻断成功。计时45分钟后去掉动脉夹,恢复血液灌注。连续缝合逐层关腹,从再灌注开始后重新计时,分别将小鼠分为30min组,1小时组,3小时组和6小时组等。在这些时间点心尖取血处死小鼠并解剖游离肝脏标本待测。褪黑素预处理方法：在缺血前15分钟腹膜下注射褪黑素(10mg/kg),之后操作同对照组。南京建成氧化指标检测试剂盒检测氧化应激指标丙二醛(MDA),髓过氧化物酶(MPO),超氧化物歧化酶(SOD)；酶联免疫法(ELISA)检测血清炎症因子核因子κB(NF-κB),肿瘤坏死因子(TNF-α),白细胞介素6(IL-6)表达量；取新鲜肝脏研磨后Anoxin VPI+FITC凋亡双染试剂盒染色流式细胞术检测肝细胞凋亡率；部分肝脏组织4%多聚甲醛固定包埋切片,进行苏木素-伊红(HE)染色和天狼星红染色,免疫组织化学方法(IHC)检测增殖细胞核抗原(PCNA)表达情况。实时定量聚合酶联反应(RT-PCR)检测NF-κB, TNF-α, IL-6,信使核糖核酸(mRNA)表达量。 结果： 天狼星红染色发现四氯化碳灌胃4周后肝硬化形成明显,胶原纤维明显增多,假小叶形成明显,对照组没有假小叶形成。肝组织羟脯氨酸含量测定发现四氯化碳灌胃组明显高于对照组,证明小鼠肝硬化模型构建成功。空白对照组,肝硬化褪黑素预处理组及肝硬化组小鼠14天生存率分别是：100%,87.5%,62.5%。肝功能指标谷草转氨酶(ALT),谷丙转氨酶(AST)明显低于对照组,炎症因子NF-KB,TNF-α表达量褪黑素预处理组明显低于照组；HE染色显示肝硬化肝脏缺血再灌后组织坏死和变性在褪黑素预处理组较对照组明显减轻。ELISA)检测血清炎症因子核因子κB(NF-κB),肿瘤坏死因子(TNF-α),白细胞介素6(IL-6)表达量在褪黑素预处理组都明显低于对照组。RT-PCR检测发现核因子NF-KB,炎症因子TNF-α, IL-6的mRNA表达在褪黑素处理组较对照组明显降低。流失细胞仪测肝细胞凋亡发现褪黑素处理组肝细胞凋亡率明显降低。增殖细胞核抗原(PCNA)染色显示肝细胞增殖在褪黑素预处理组没有明显增加。 结论： 褪黑素能够提高肝硬化小鼠缺血再灌注后生存率；能够保护肝功能：能够减轻氧化应激损伤；能够减少肝细胞凋亡率；可能是通过清除活性氧分子(ROS)减少激活炎症通路,炎症因NF-κ B及其下游通路炎症因子TNF-α,IL-6的表达和激活；使缺血再灌注过程中NF-κ B的双重调控作用倾向于减轻过度表达的炎症因子对肝硬化肝脏的损伤作用,促进肝细胞增殖的作用并不明显。
[Abstract]:Objective:
The purpose of this study is to study the protective effect of melatonin on liver ischemia reperfusion injury in liver cirrhosis mice, the effect of melatonin on the survival rate of liver cirrhosis after ischemia-reperfusion injury, the effect on liver function, the effect on the apoptosis rate, the influence on oxidative stress, and the expression of inflammatory factors, and the analysis of inflammatory factors. To investigate the relationship between melatonin and ischemia-reperfusion injury, and explore the possibility of melatonin as a protective agent for ischemia-reperfusion injury.
Background:
The study showed that more than half of the patients with liver cancer were associated with cirrhosis and the liver function reserve decreased. Compared with the normal liver, the liver was more intolerant of the ischemic reperfusion injury in the liver, the incidence of liver failure in the perioperative period and the mortality rate of the patients were higher than that in the normal liver. The main original of liver injury caused by ischemia reperfusion was the primary liver injury. The direct damage to liver tissue caused by a large number of active oxygen molecules produced by adenosine triphosphate metabolites in the process of reperfusion and the activation of the inflammatory pathway by active oxygen molecules lead to the release of a large number of inflammatory factors leading to the apoptosis and necrosis of the liver cells. Reactive oxygen molecules can reduce the damage to liver tissue caused by ischemia-reperfusion. Melatonin is a small molecular substance that can be secreted by many organs such as the pineal body and the liver. It has the role of regulating rhythmic physiological activity. Previous studies emphasized the effect of melatonin on the central nervous system and the antioxidant energy of melatonin in recent years. Melatonin has strong antioxidant capacity, with small molecular weight, high fat solubility, wide distribution, wide spectrum antioxidant, low toxicity and high safety. It can remove oxygen free radicals and reduce normal liver ischemia-reperfusion injury. However, there are certain inflammatory molecules expression in the liver of liver cirrhosis, blood distribution is the same as normal. The liver is different, and the level of cell metabolism is different from that of normal liver. There are few studies on liver ischemia reperfusion injury in liver cirrhosis. The purpose of this study is to investigate whether melatonin preconditioning can reduce the effect of liver ischemia reperfusion injury in liver cirrhosis and explore the specific mechanism.
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