【作者】 卢静；

【导师】 郑世军；

【作者基本信息】 中国农业大学 ， 预防兽医， 2013， 博士

【摘要】 肝病是威胁我国人民健康的重大疾病之一,其患病率和致死率均很高。肝损伤动物模型在肝病致病机理研究及药物研发中非常重要。肝病种类很多,但大多会引起肝脏炎症,最终导致不同程度的肝损伤。四氯化碳是常见的制作肝损伤动物模型的化学药物,由于于其成模率高、重复性好,四氯化碳急、慢性肝损伤模型在研究肝脏疾病中得以广泛应用。然而四氯化碳对人体危害大,因此探寻对人危害性小、安全性高的化合物来制作肝损伤动物模型非常迫切需要。研究发现D氨基半乳糖肝损伤大鼠模型具有成模率高、重复性好、对人体危害性低等优点。但以D氨基半乳糖诱导小鼠肝损伤模型尚未见报道。本研究通过D氨基半乳糖和低浓度四氯化碳分别建立急、慢性肝损伤小鼠动物模型,利用组织学苏木精—伊红染色(HE)和Masson染色观察小鼠肝组织结构变化、细胞形态及纤维化程度,利用常规生化方法检测血清天冬氨酸转氨酶(AST)和谷丙转氨酶(ALT)的浓度确定肝组织损伤程度,对肝损伤小鼠模型进行评价。研究结果表明,D氨基半乳糖以10μl/g的剂量,通过腹腔注射,1次/2天,连续注射8周的小鼠,能引起稳定且明显的肝组织损伤。该结果证明应用D氨基半乳糖能成功诱导出小鼠肝损伤模型。该模型的建立为深入研究肝脏疾病形成机理和筛选肝病治疗性药物提供有效的手段,具有重要科学意义和应用价值。低浓度四氯化碳以10μl/g的剂量,通过腹腔注射,1次/3天,连续注射8周的小鼠,能引起稳定且明显的肝组织损伤。我们应用小鼠肝损伤动物模型进行了应激对肝损伤影响的深入研究,为揭示应激与肝损伤的关系提供参考。应激是机体受到刺激后产生的非特异性适应反应,是一种生理和心理对环境的抵抗状态,以维护机体的稳定状态。束缚应激作为一种非损伤性刺激,能够较好地模拟人类生活中“无法控制”的拥挤和适当强度的生活、工作等状态,尤其是模拟宇航员在宇宙飞行中的应激变化,然而,束缚应激对在急、慢性肝损伤是否有影响目前尚不清楚。本文通过对急、慢性肝损伤小鼠模型施加定时定量的束缚应激,利用HE和Masson染色观察小鼠肝组织结构、细胞形态及纤维化程度,并利用常规生化方法检测血清AST和ALT的浓度,分析束缚应激对肝损伤的影响。研究结果表明束缚应激能减轻小鼠肝细胞的损伤和肝纤维化的形成,显著降低肝损伤小鼠血清AST和ALT水平。此外,利用酶联免疫分析(ELISA)检测肝组织中的P450酶、超氧化物歧化酶(SOD)和脂质过氧化终产物丙二醛(MDA)的浓度,发现束缚应激后小鼠肝脏内的P450酶和MDA水平显著降低,与之相反,SOD水平显著增高。这些结果说明束缚应激可能通过降低细胞内氧化产物的水平,从而降低氧化应激对肝细胞造成的损伤。因此,该研究结果从细胞氧化代谢的途径阐明了束缚应激对肝损伤缓解作用的机制,为深入研发治疗非创伤性肝脏疾病药物提供了新的思路,也为航天医学提供参考。更多还原

【Abstract】 Liver disease is one of the major diseases threatening the health of people in China because of its high morbidity and mortality. Liver injury animal model is very important in the study of pathogenesis of liver disease and drug development. There are many types of liver diseases, but most of them can cause liver inflammation and eventually lead to varying degrees of hepatic injury. Carbon tetrachloride is the common chemical for production of hepatic injury animal model, the carbon tetrachloride induced acute and chronic liver injury models were widely used in the study of liver diseases because of its high success rate and good repeatability of modeling. However, carbon tetrachloride is high toxic for human, therefore, to explore less toxic and high safety of chemicals for producation of liver injury animal model is very urgent. Previous studies showed that D-galactosamine liver injury model in rat also has a high success rate and good repeatability of modeling, and importantly, D-galactosamine is far less toxic for human than carbon tetrachloride. However, the D-galactosamine-induced liver injury model in mouse has not been reported yet. In this study, we established the acute and chronic liver injury mouse models using D-galactosamine and carbon tetrachloride. We observed the hepatic tissue change, cell morphology and fibrosis using hematoxylin-eosin staining (HE) and masson staining assays, and measured the levels of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) using the conventional biochemical methods. We evaluated the liver injury mouse models through the analysis of liver pathological morphology and serum biochemical indices. The results show that D-galactosamine induced liver injury in mouse model was successfully established by intraperitoneal injection. The model could be a new choice for the study of liver diseases and drug development.Stress is a nonspecific adaptive response when the body was stimulated.It is a physiological and psychological state of confrontation with the environment in order to maintain homeostasis. Restraint stress,as a non-invasive stimulation,can well simulate human states such as "out of control" crowded, frustration and other daily challenging conditions, especially it can mimic working state in the space shuttle. However, it is still not clear whether restraint stress during acute or chronic liver injury can improve the resistant ability of body and reduce the degree of liver injury. We employed the restraint stress quantitatively on theacute and chronic liver injury mouse models regularly, observed the hepatic lobules, cell morphology and fibrosis using the HE and masson stained liver tissue slides, and measured the levels of serum AST and ALT using the conventional biochemical methods, and then analyzed the effects of restraint stress on liver injury. The results show that restraint stress reduced liver cell damage and liver fibrosis, and significantly decreased the serum AST and ALT levels. These results suggest that restraint stress has a protective effect on liver injury. In addition, we measured the levels of P450enzymes,super oxide dismutase(SOD) and lipid peroxidation end product malondialdehyde(MDA) in liver homogenates using the enzyme-linked immunosorbent assay(ELISA).We found that the levels of P450enzymes and MDA significantly decreased after restraint stress.On the contrary, the SOD levels were significantly increased.These results suggest restraint stress might reduce the levels of oxidation products, thereby inhibiting the oxidative stress-induced damage to the liver cells. These results will be of great help to explore the mechanism under lying protective mechanism of restraint stress on liver injury via oxidative stress pathway and will be also helpful to the development of new drugs for the treatment of non-invisive liver injury and provide reference data for Space medicine.更多还原