發(fā)布時(shí)間：2018-05-25 10:04

  本文選題：褪黑素 + 肝硬化�。� 參考：《第二軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：目的： 本研究目的在于研究褪黑素對(duì)肝硬化小鼠肝臟缺血再灌注損傷的保護(hù)作用,研究褪黑素對(duì)肝硬化缺血再灌注損傷后生存率的影響、對(duì)肝功能的影響、對(duì)細(xì)胞凋亡率的影響、對(duì)氧化應(yīng)激的影響、以及對(duì)炎癥因子表達(dá)的影響；分析炎癥因子表達(dá)同缺血再灌注損傷的關(guān)系,探討褪黑素作為缺血再灌注損傷保護(hù)劑的可能。 背景： 研究顯示肝癌患者中半數(shù)以上合并有肝硬化,肝功能儲(chǔ)備下降。與正常肝臟相比,肝硬化背景下的肝臟對(duì)術(shù)中缺血再灌注損傷更加不耐受,圍手術(shù)期肝功能衰竭發(fā)生率及患者死亡率高于正常肝臟人群。缺血再灌注造成肝臟傷害的主要原因包括再灌注過程中三磷酸腺苷代謝產(chǎn)物產(chǎn)生的大量活性氧分子對(duì)肝組織造成的直接損傷,以及活性氧分子激活炎癥通路,導(dǎo)致大量炎癥因子釋放,導(dǎo)致肝細(xì)胞凋亡壞死。如果能夠在缺血再灌注之前預(yù)防性的使用氧自由基清除劑,清除活性氧分子則可以減輕缺血再灌注對(duì)肝組織造成的損傷。褪黑素是有松果體及肝臟等多個(gè)器官都可以分泌的小分子物質(zhì),具有調(diào)節(jié)機(jī)體節(jié)律性生理活動(dòng)的作用,以往的研究強(qiáng)調(diào)褪黑素對(duì)中樞神經(jīng)系統(tǒng)的作用,近年來褪黑素的抗氧化能力得到重視。褪黑素具有強(qiáng)大的抗氧化能力,具有分子量小,脂溶性高,分布廣泛,廣譜抗氧化,毒性小安全性高等優(yōu)點(diǎn),能夠清除氧自由基,減輕正常肝臟缺血再灌注損傷。但是肝硬化肝臟中本身存在一定的炎癥分子表達(dá),血液分布同正常肝臟也有不同,細(xì)胞代謝水平同正常肝臟也有區(qū)別,目前對(duì)肝硬化肝臟缺血再灌注損傷的研究較少。本研究目的在于探討證實(shí)褪黑素預(yù)處理是否具有減輕肝硬化肝臟缺血再灌注損傷作用,并進(jìn)一步探討其具體機(jī)制。 方法： 100只BALB/C小鼠隨機(jī)分為四氯化碳(CCL4)灌胃誘導(dǎo)肝硬化組和正常肝對(duì)照組,肝硬化組又分為肝硬化缺血再灌注褪黑素預(yù)處理組和肝硬化缺血再灌注對(duì)照組。生存率檢測(cè)分三大組,分別是空白組,肝硬化組和肝硬化褪黑素預(yù)處理組。外科手術(shù)過程為4%水合氯醛麻醉后,逐層開腹,兩根無菌脫脂棉簽暴露肝門,動(dòng)脈夾選擇性夾閉門靜脈左支即選擇性阻斷小鼠肝中葉及肝左葉入肝血流,約5秒鐘后肝中葉及左葉顏色迅速變暗,提示阻斷成功。計(jì)時(shí)45分鐘后去掉動(dòng)脈夾,恢復(fù)血液灌注。連續(xù)縫合逐層關(guān)腹,從再灌注開始后重新計(jì)時(shí),分別將小鼠分為30min組,1小時(shí)組,3小時(shí)組和6小時(shí)組等。在這些時(shí)間點(diǎn)心尖取血處死小鼠并解剖游離肝臟標(biāo)本待測(cè)。褪黑素預(yù)處理方法：在缺血前15分鐘腹膜下注射褪黑素(10mg/kg),之后操作同對(duì)照組。南京建成氧化指標(biāo)檢測(cè)試劑盒檢測(cè)氧化應(yīng)激指標(biāo)丙二醛(MDA),髓過氧化物酶(MPO),超氧化物歧化酶(SOD)；酶聯(lián)免疫法(ELISA)檢測(cè)血清炎癥因子核因子κB(NF-κB),腫瘤壞死因子(TNF-α),白細(xì)胞介素6(IL-6)表達(dá)量；取新鮮肝臟研磨后Anoxin VPI+FITC凋亡雙染試劑盒染色流式細(xì)胞術(shù)檢測(cè)肝細(xì)胞凋亡率；部分肝臟組織4%多聚甲醛固定包埋切片,進(jìn)行蘇木素-伊紅(HE)染色和天狼星紅染色,免疫組織化學(xué)方法(IHC)檢測(cè)增殖細(xì)胞核抗原(PCNA)表達(dá)情況。實(shí)時(shí)定量聚合酶聯(lián)反應(yīng)(RT-PCR)檢測(cè)NF-κB, TNF-α, IL-6,信使核糖核酸(mRNA)表達(dá)量。 結(jié)果： 天狼星紅染色發(fā)現(xiàn)四氯化碳灌胃4周后肝硬化形成明顯,膠原纖維明顯增多,假小葉形成明顯,對(duì)照組沒有假小葉形成。肝組織羥脯氨酸含量測(cè)定發(fā)現(xiàn)四氯化碳灌胃組明顯高于對(duì)照組,證明小鼠肝硬化模型構(gòu)建成功。空白對(duì)照組,肝硬化褪黑素預(yù)處理組及肝硬化組小鼠14天生存率分別是：100%,87.5%,62.5%。肝功能指標(biāo)谷草轉(zhuǎn)氨酶(ALT),谷丙轉(zhuǎn)氨酶(AST)明顯低于對(duì)照組,炎癥因子NF-KB,TNF-α表達(dá)量褪黑素預(yù)處理組明顯低于照組；HE染色顯示肝硬化肝臟缺血再灌后組織壞死和變性在褪黑素預(yù)處理組較對(duì)照組明顯減輕。ELISA)檢測(cè)血清炎癥因子核因子κB(NF-κB),腫瘤壞死因子(TNF-α),白細(xì)胞介素6(IL-6)表達(dá)量在褪黑素預(yù)處理組都明顯低于對(duì)照組。RT-PCR檢測(cè)發(fā)現(xiàn)核因子NF-KB,炎癥因子TNF-α, IL-6的mRNA表達(dá)在褪黑素處理組較對(duì)照組明顯降低。流失細(xì)胞儀測(cè)肝細(xì)胞凋亡發(fā)現(xiàn)褪黑素處理組肝細(xì)胞凋亡率明顯降低。增殖細(xì)胞核抗原(PCNA)染色顯示肝細(xì)胞增殖在褪黑素預(yù)處理組沒有明顯增加。 結(jié)論： 褪黑素能夠提高肝硬化小鼠缺血再灌注后生存率；能夠保護(hù)肝功能：能夠減輕氧化應(yīng)激損傷；能夠減少肝細(xì)胞凋亡率；可能是通過清除活性氧分子(ROS)減少激活炎癥通路,炎癥因NF-κ B及其下游通路炎癥因子TNF-α,IL-6的表達(dá)和激活；使缺血再灌注過程中NF-κ B的雙重調(diào)控作用傾向于減輕過度表達(dá)的炎癥因子對(duì)肝硬化肝臟的損傷作用,促進(jìn)肝細(xì)胞增殖的作用并不明顯。
[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.
Method錛，

本文編號(hào)：1933024