【作者】 田苗；

【导师】 李春岩； 宋学琴；

【作者基本信息】 河北医科大学 ， 神经病学， 2008， 硕士

【摘要】 目的:1993年,Rosen DR等发现,部分家族性肌萎缩侧索硬化(fALS)的发病与编码铜/锌超氧化物歧化酶(Cu/Zn SOD)的SOD1基因突变有关。该发现为我们提供了一个明确的运动神经元死亡的原因。到目前为止,已确定在20%fALS和4%的散发性肌萎缩侧索硬化(sALS)中有SOD1基因突变,其突变数目已超过100种。其中,93位的甘氨酸(Gly)突变为丙氨酸(Ala)为常见的突变之一。转染了人突变SOD1基因的转基因小鼠具有与ALS病人相同的临床表现,现已成为国际公认的研究ALS的理想模型。在ALS病人及转基因动物模型内均存在蛋白质、脂质和核酸这些生物大分子氧化损伤的证据,提示氧化应激是疾病进程中的一个重要方面,但是机体自身的抗氧化系统是否在疾病的进展过程中起到了保护作用,是否随疾病的进展发挥更高的保护作用,我们不清楚。目前文献报道Nrf2/ARE信号通路的激活可在多种组织中诱导一系列内源性的细胞保护基因上调,其中包括抗氧化酶、抗氧化蛋白、抗炎和解毒的蛋白,它们的活化可以对机体起到保护作用。本试验目的:探讨不同时期B6SJL-TgN(SOD1G93A)1Gur转基因小鼠(携带人突变SOD1基因)与同窝野生型小鼠和B6SJL-TgN(SOD1)2Gur转基因小鼠(携带正常人SOD1基因)相比,脊髓组织中是否存在Nrf2和抗氧化酶HO-1、NQO1表达的差异,了解不同时期机体抗氧化能力的变化情况,也为深入研究转基因小鼠中Nrf2/ARE通路提供前期基础。方法:1、转基因鼠的繁殖所有动物均饲养在恒温(25-27℃)、恒湿和无菌条件的(Specific pathogen free, SPF)环境中,喂以灭菌的SPF级颗粒型鼠类饲料。为维持B6SJL-TgN (SOD1G93A)1 Gur转基因小鼠突变基因稳定下传,将B6SJL SOD1G93A/+半合子雄鼠与B6SJLF1/J半合子雌鼠交配繁殖.子代鼠经基因鉴定确定是否带有人突变的基因2、实验分组研究对象共分为三个时期:症状前期(80天)、症状早期(120天)、终末期。每个时期组除了选择SOD1G93A小鼠及同窝野生型小鼠外,还选取一组同龄hSOD1小鼠作对照,为排除小鼠脊髓中人超氧化物岐化酶表达增多对实验结果的干扰。3、Nrf-2蛋白水平的测定提取脊髓组织的浆蛋白,用Synergy-HT多功能酶标仪测量OD数值,可检测样本蛋白浓度,计算100蛋白上样量。走SDS-PAGE电泳,100V 4度转膜2小时、5%脱脂奶粉封闭1小时;一抗孵育,4oC,过夜;TPBS震荡漂洗5min×5次;荧光二抗孵育,室温,2h;TPBS震荡漂洗5min×5次;Odyssey Infrared Imaging System检测分析。4、HO-1、NQO1基因表达水平的测定取脊髓组织6mg,提取总RNA。用Synergy-HT多功能酶标仪测量OD260/O Odyssey Infrared Imaging System检测分析。D280比值,可检测RNA的纯度和含量,选用OD260/OD280比值为1.8-2.0的RNA用作反转录,合成cDNA。扩增HO-1、NQO1、nrf-2目的基因,在1.5%琼脂糖凝胶(含Goldview染料)上电泳,用GBOX-HR全自动凝胶成像系统拍摄打印实验结果,用凝胶图像分析系统分析结果。结果:与两组对照组相比,转基因小鼠在发病前期脊髓中Nrf-2蛋白水平及抗氧化酶HO-1、NQO1的基因表达无明显变化;症状期、终末期转基因小鼠脊髓中Nrf-2蛋白水平下降及抗氧化酶HO-1、NQO1基因表达明显升高。结论:本实验在肌萎缩侧索硬化转基因小鼠模型的基础上,发现疾病症状早期和终末期存在着Nrf-2激活及下游抗氧化酶基因表达上调的现象。但这些酶的含量很低,对机体的保护作用还需进一步研究。更多还原

【Abstract】 Objectives: In 1993, Rosen DR found that some of the familial amyotrophic lateral sclerosis have mutation of gene coding copper / zinc superoxide dismutase (Cu / Zn SOD) of the SOD1. The discovery provides us with a clear reason for motor neuron death. So far, 20 percent of fALS and 4% Sporadic amyotrophic lateral sclerosis (sALS) have the SOD1 gene mutation and more than 100 kinds of mutation have been identified. Among them, glycine (Gly) mutation for alanine (Ala) located site 93 in the gene is the most common. Transgenic mice transfected with mutant SOD1 gene have the same clinical manifestations with ALS patients,which has been internationally recognized as the ideal model for ALS research.In ALS patients and transgenic animal models, there are proof of oxidative damage of some biological macromolecules such as proteins, lipids and nucleic acid. These suggests that oxidative stress involve in the pathogenesis of the disease. whether the body’s own antioxidant system plays a protective role in the disease or play a more protective role in the progress of the disease is not sure. Some researchers reported that the activation of Nrf2/ARE signaling pathway can induce an endogenous increase including the antioxidant enzymes, antioxidant proteins, anti-inflammatory and detoxification proteins, all of these can play a protective role. This study will investigate the expression of antioxidant enzymes Nrf2 and HO-1, NQO1 in the spinal cord of B6SJL-TgN (SOD1G93A) 1Gur transgenic mice (Carry mutant SOD1 gene) during the different periods of life and help us to understand the oxidation resistance ability and the Nrf2/ARE pathway in ALS transgenic mouse.Methods:1 the propagation of transgenic miceAll animals were kept in constant temperature (25-27℃), hang wet and sterile conditions (Specific pathogen free SPF) environment and fed with the sterilization of SPF rodents feed particles. To maintain B6SJL-TgN (SOD1G93A) 1 Gur transgenic mice under-gene mutation, B6SJL SOD1G93A / + hemizygous males and B6SJLF1 / J hemizygous females were mated , the genetic offspring mice were identified with a mutant gene determination .2 the experimental grouptheSOD1G93Amice were divided into three groups Symptomless-stage (80 days), early symptoms (120 days), end-stage. wild-type mice and hSOD1 were the control, hSOD1mice were used to exclude the influence of human SOD.3 Nrf-2 protein level determinationplasma proteins were extracted from Spinal cord tissue and measured by Synergy-HT-Meibiaoyi OD numerical measu- rements, protein concentration , protein of 100 on the sample volume. Take SDS-PAGE electrophoresis, 100 V 4℃to film two hours, 5% skim milk closed one hour; an anti-incubation, 4℃, overnight; TPBS concussion rinsing 5 min×5; fluorescence two-incubated at room temperature for 2 h; TPBS concussion Rinse 5 min×5; Odyssey Infrared Imaging System Analysis. 4 HO-1, NQO1 gene expression level determinationSpinal cord of 6 mg was isolated for total RNA extraction. OD values were measured by Synergy HT-by-Meibiaoyi measurement. 1.8-2.0 of OD260/OD280 ratio were selected for reverse transcription of RNA and cDNA synthesis. HO-1, NQO1, nrf-2 gene were amplified and took electrophoresis in 1.5% agarose gel (containing Goldview dye). Theresults were printed by Automatic GBOX-HR gel imaging systems and analyzed by the gel image analysis Analysis system.Results: Compared with the two control groups, the Nrf-2 protein level and antioxidase HO-1, the NQO1 gene expression of spinal cord from the ALS transgene mouse in Symptomless-stage were not changed; while the Nrf-2 protein level decreased and antioxidase HO-1, the NQO1 gene expression increased during the symptom-stage and end-stage.Conclusion: The Experiment was studied in amyotrophic lateral sclerosis transgenic mouse model-ALS SOD1G93A mice. During the early stage and end-stage of diseases, there are activation of Nrf-2 and increase expression of downstream antioxidant enzymes including HO-1 and NQO1 in the spinal cord of transgenic mice. However, the protective effect of these enzymes needs further study.更多还原