【作者】 姜巍；

【导师】 陈勇；

【作者基本信息】 第四军医大学 ， 外科学， 2010， 硕士

【摘要】 多种刺激包括缺氧、缺糖、病毒感染等都可以导致内质网应激。近年来很多证据表明内质网应激和许多疾病密切相关。内质网应激触发的未折叠蛋白反应通过关闭蛋白翻译、促进分子伴侣表达、促进错误折叠蛋白的转运与降解等机制降低内质网负荷,提示内质网应激预处理具有潜在的治疗作用。本实验通过建立了内质网应激预处理条件下的大鼠肝脏缺血再灌注损伤模型,探讨内质网应激预处理在体内的应用。1.以衣霉素为内质网应激诱导剂,采用肝脏70%缺血再灌注损伤模型。动物分组:100μg/kg体重-4天、100μg/kg体重-5天、100μg/kg体重-6天、50μg/kg体重-5天、300μg/kg体重-5天、对照组,观察不同诱导时间与给药剂量情况下,术后血清转氨酶水平的变化。结果:给药100μg/kg体重诱导5天,大鼠术后转氨酶水平显著低于对照组。其它组与对照组无统计学差异。2.将雄性Wistar大鼠分为4组:安慰剂-假手术组(A组)、预处理-假手术组(B组)、安慰剂-IR组(C组)、预处理-IR组(D组),检测各组动物术前及术后的血清转氨酶含量,肝脏形态学改变及肝组织GRP78的表达情况。结果:B组GRP78表达水平高于A组,提示衣霉素在体内成功诱导了内质网应激。D组术后转氨酶水平显著低于C组,病理切片观察D组肝脏组织损伤情况好于C组。在电镜下D组内质网结构较C组完整。结论:本研究发现在特定的诱导时间与剂量条件下,衣霉素诱导的内质网应激预处理对大鼠肝脏缺血再灌注损伤有显著的保护作用,提示内质网应激预处理可能是肝脏外科新的治疗手段。更多还原

【Abstract】 The accumulation of unfolded proteins in the endoplasmic reticulum (ER) represents a cellular stress induced by multiple stimuli and pathological conditions. These include hypoxia, oxidative injury, high-fat diet, hypoglycemia, protein inclusion bodies and viral infection. ER stress triggers an evolutionarily conserved series of signal transduction events, which constitutes the unfolded protein response. Resent reports showed that ER stress is associate with a lot of diseases. The initial intent of the UPR is to reestablish homeostasis and normal ER function, and adaptive mechanisms predominantly involve activation of transcriptional programs that induce expression of genes that are capable of enhancing the protein folding capacity of the ER and genes for ER-assisted degradation (ERAD). These adaptive mechanisms suggest the possibility of therapeutic approaches targeting ER stress in IR injury. In this study we established a rat IR injury model under preconditioning with ER stress and investigated the potential of therapeutic approaches targeting ER stress in IR injury in rats. 1. Establishing animal model with IR injury under ER stress preconditioning. SD rats were pretreated with the ER stress inducer tunicamycin (TM) before IR operation. For evaluating the protective effect of preconditioning with ER stress, animals were divided into a control group and five pretreated groups. Different dose of TM (50μg/kg body wt, 100μg/kg body wt, 300μg/kg body wt) and different inducing time (4d, 5d, 6d) were progressed in this study. Partial hepatic IR injury model (45 min of ischemia via vascular clamping and 24 h reperfusion) were established and the severity of liver damage was determined by measuring serum ALT and AST levels. At the dose of 100μg/kg body wt and 5d before operation, preconditioning ameliorated the IR injury significantly (serum ALT at 6h after reperfusion 1506.28±401.98 u/L versus 2671.33±565.53 u/L, serum AST at 6h after reperfusion 3139.71±989.31 versus 5949.83±1205.51 u/L, serum ALT at 12h after reperfusion 723.71±278.56u/L versus 1141.33±231.63u/L, serum AST 2562.00±1673.47 versus 2996.83±882.68u/L, p<0.05 respectively). But no protection occurred in other groups.2. Verifying the protective effect of ER stress preconditioning. Male wistar rats were divided into control-sham group (A), TM-sham group (B), control-IR group (C) and TM-IR group (D). TM preconditioning in rats showed significantly protection against IR injury evidenced by lack alteration of serum ALT (109.14±22.26 versus 513.72±304.90 u/L p<0.05) and normal liver histology. Immunohistochemistry and Western blotting showed over expression of the ER stress–inducible chaperones glucose-regulated protein 78 (GRP78) in IR treated rats. High expression of GRP78 in TM-sham group compared with control-sham group implied that ER stress was induced by TM administration. More abundant ER was observed in preconditioning group compared with control group under transmission electron microscope (TEM) when IR injury occurred.Conclusion: Preconditioning with ER stress ameliorates the severity of IR injury in certain condition. These findings suggest the possibility therapeutic approaches targeting ER stress in hepatic IR injury.更多还原