SOD1-G93A小鼠运动皮层与腰髓中SIRT1的变化及白藜芦醇对其的影响

发布时间：2018-05-25 03:15

本文选题：肌萎缩侧索硬化 + 长寿蛋白　；参考：《河北医科大学》2014年硕士论文

【摘要】：目的：肌萎缩侧索硬化（Amyotrophic lateral sclerosis,ALS）是一种同时累及上、下运动神经元，及其支配的躯干、四肢和头面部肌肉的慢性进行性变性疾病。临床上常表现为上、下运动神经元合并受损的混合性瘫痪。该病自发现以来已有100多年的历史，虽经大量研究，其发病机理仍不清楚，仍无有效的治疗方法，病人的生活质量差，平均存活期短，仅有3-5年。目前关于ALS发病机制的假说有很多，比如兴奋性氨基酸毒性、氧化应激、免疫学机制、线粒体功能障碍和异常蛋白的聚集等。然而，选择性运动神经元丢失的根本原因依然不详，目前仍无特效治疗方法。 SIRT1（sirtuins）又名长寿蛋白，最早发现于酵母菌中，是一类从细菌到人类都高度保守的NAD+依赖的去乙酰化蛋白酶类，它分七个亚型，分别为SIRT1-SIRT7,研究最多的是SIRT1，它与酵母菌的SIR2基因的同源性最高，可以使组蛋白去乙酰化，调节许多蛋白酶类的活性，还能从基因水平影响蛋白的表达，参与脂肪代谢、能量限制、免疫功能、炎症反应、自噬、增殖与老化、细胞凋亡、肿瘤等生理功能。近年来，人们对sirtuins在2型糖尿病、癌症、心血管疾病等多种疾病和其相关并发症、遗传性疾病和神经退行性变疾病中的研究日益增多。作为一种保护性蛋白，在朊蛋白病模型的研究中，SIRT1可以通过保护线粒体来维持细胞生存；帕金森病（PD）模型中，SIRT1可以对抗氧化应激损伤；阿尔茨海默病（AD）中，有研究指出SIRT1可以减少β淀粉样蛋白前体（APP）形成，减少β淀粉样蛋白沉积。白藜芦醇（Resveratrol，Res）是多酚类化合物，已被证实可以激活SIRT1并且广泛用于各种实验中，是近来研究较多的SIRT1的激动剂，它主要来源于蓼科植物的根茎中。白藜芦醇具有抗炎、抗癌、抗氧化、抗心血管疾病等作用，近来对于白藜芦醇在神经退行性疾病中有益作用的研究日益增多。然而，在同样作为神经变性疾病的ALS中，SIRT1的表达有无变化，有学者证实SIRT1在ALS中表达是增多的，但也有学者证实SIRT1是减少的，目前其在ALS中的作用及表达变化还未有定论，我们所感兴趣和要研究的是随着疾病的进展，SIRT1在ALS小鼠运动皮层与腰髓中会有怎样的动态变化以及给予白藜芦醇后对SIRT1的表达量会产生怎样的影响。 SOD1-G93A转基因小鼠是目前研究ALS较为理想的动物模型之一，本实验旨在观察随着疾病的进展，SOD1-G93A转基因小鼠运动皮层及腰髓中SIRT1的动态变化，同时给予SIRT1激活剂白藜芦醇后SOD1-G93A转基因小鼠运动皮层与腰髓中SIRT1的变化，来进一步加深对ALS的研究，探讨SIRT1成为ALS的治疗靶点是否具有可行性和白藜芦醇在ALS中的作用。方法：选取SOD1-G93A转基因小鼠为实验组，根据病程分为症状前期（60天）组、症状早期（Onset）组、终末期(End)组，选取90天的野生型小鼠作为对照组，各组3只；白藜芦醇（Sigma，101237259）0.375g，溶于7.5ml无水乙醇中，加生理盐水定容至50ml,配成7.5mg/ml的溶液，同时配制15%的乙醇生理盐水溶液作为溶剂；处理组分3组，选取70dSOD1-G93A转基因小鼠，分为空白组、溶剂组、白藜芦醇组，，每组根据给药时间分2个亚组，每组6只，从70d（症状前期）开始分别给予上述白藜芦醇溶液30mg/kg/d胃内灌注（白藜芦醇组）、同等体积的溶剂胃内灌注（溶剂组）、不给予任何处理（空白组），分别连续灌注至症状早期（连续给药20-30天）、终末期（连续给药50-60天）后取材，以10%水合氯醛(350mg/Kg体重)腹腔注射麻醉，处死小鼠，取运动皮层与腰髓迅速投入液氮冷冻，之后保存于-80℃冰箱；4%多聚甲醛经心脏灌注，之后剥离小鼠运动皮层与腰髓，用4%多聚甲醛浸泡或用2.5%戊二醛固定组织，利用Western blot技术和免疫组织化学技术观察SOD1-G93A转基因小鼠运动皮层与腰髓中SIRT1的动态表达情况及给予白藜芦醇后SOD1-G93A转基因小鼠运动皮层及腰髓中SIRT1的变化。并利用spss13.0对数据进行分析。结果：1SOD1-G93A转基因小鼠运动皮层与腰髓中随疾病发展SIRT1的动态变化：Wstern blot技术检测SOD1-G93A转基因小鼠运动皮层与腰髓SIRT1表达结果显示，与对照组相比，60d组SIRT1表达量无明显变化，症状早期及终末期组SIRT1表达量均增加。免疫组织化学染色可见症状早期组、终末期组SIRT1表达增加，终末期腰髓中运动神经元明显减少。 2给予白藜芦醇后SOD1-G93A转基因小鼠运动皮层与腰髓中SIRT1的表达量变化：Wstern blot示给予白藜芦醇后，症状早期运动皮层及腰髓中白藜芦醇组SIRT1表达量较溶剂组降低，较空白组无明显变化；症状早期溶剂组SIRT1表达量较空白组增多。终末期各组之间SIRT1表达量均无明显变化。免疫组织化学染色显示症状早期SOD1-G93A转基因小鼠运动皮层与腰髓中白藜芦醇组较溶剂组SIRT1表达减少；白藜芦醇组SIRT1表达较空白组有所增加，但无明显变化；溶剂组较空白组SIRT1表达明显增加。终末期三组间SIRT1表达无明显差异。结论：随着疾病的进展，SOD1-G93A转基因小鼠运动皮层与腰髓中SIRT1的表达量逐渐增加，而当症状早期给予白藜芦醇后，SIRT1的表达量较空白组无明显变化，较溶剂组减少，而给予溶剂组SIRT1表达量较白藜芦醇组及空白组均有明显增高，终末期各组之间均无明显变化，这说明白藜芦醇没有起到激活SIRT1的作用，甚至起到了降低SRT1的作用，白藜芦醇对于ALS未起到预期的保护效应，其在ALS模型中的保护作用有限，白藜芦醇的作用机制尚不能定论，需进一步深入研究，同时对ALS的研究起到一定参考作用。
[Abstract]:Objective: amyotrophic lateral sclerosis (Amyotrophic lateral sclerosis, ALS) is a chronic progressive disease involving both upper and lower motor neurons and its dominant trunk, extremities, and head and face muscles. It is often manifested in the upper and lower motor neurons with damaged mixed paralysis. The disease has been found for more than 100 years since it was discovered. History, although a lot of research, its pathogenesis is still unclear, there is still no effective treatment, the patient's quality of life is poor, the average survival time is short, only 3-5 years. At present, there are many hypotheses about the pathogenesis of ALS, such as excitatory amino acid toxicity, oxidative stress, immunological mechanism, mitochondrial dysfunction and abnormal protein aggregation. The underlying reason for the loss of selective motor neurons is still unknown.
SIRT1 (sirtuins), also known as longevity protein, is first found in yeast. It is a class of NAD+ dependent deacetylation proteases from bacteria to human beings. It is divided into seven subtypes, SIRT1-SIRT7, and SIRT1. It has the highest homology with the SIR2 gene of yeast, which can make histone deacetylation and regulate many of them. The activity of protease can also affect the expression of protein from gene level, participate in lipid metabolism, energy restriction, immune function, inflammatory response, autophagy, proliferation and aging, cell apoptosis, tumor and other physiological functions. In recent years, people have inherited diseases and related complications of sirtuins in type 2 diabetes, cancer, cardiovascular disease, and its related complications, hereditary diseases As a protective protein, in the study of the prion disease model, SIRT1 can maintain the survival of the cells by protecting the mitochondria as a protective protein; in the Parkinson disease (PD) model, SIRT1 can combat oxidative stress damage; in Alzheimer's disease (AD), research indicates that SIRT1 can reduce the accumulation of beta amylin. Amyloid precursor protein (APP) is formed to reduce beta amyloid deposition.
Resveratrol (Res) is a polyphenol compound. It has been proved to be able to activate SIRT1 and widely used in various experiments. It is the most recent agonist of SIRT1. It is mainly derived from the rhizomes of Polygonum plants. Resveratrol has anti-inflammatory, anticancer, antioxygenation, anti cardiovascular disease and so on. Recently, resveratrol is in the God of resveratrol Research on the beneficial effects of degenerative diseases is increasing.
However, in the same ALS as neurodegenerative disease, there is a change in the expression of SIRT1. Some scholars have confirmed that the expression of SIRT1 in ALS is increased, but some scholars have confirmed that SIRT1 is reduced, and its role and expression in ALS are not yet determined. We are interested in and should study with the progression of the disease, SIRT1 in ALS mice What dynamic changes in the motor cortex and lumbar spinal cord and the effect of resveratrol on the expression of SIRT1 will be affected.
SOD1-G93A transgenic mice are one of the most ideal animal models for studying ALS. This experiment aims to observe the dynamic changes of SIRT1 in the motor cortex and lumbar spinal cord of SOD1-G93A transgenic mice, and to give the changes of the motor cortex and SIRT1 in the lumbar spinal cord of the SOD1-G93A transgenic mice after the SIRT1 activator of resveratrol. One step is to deepen the study of ALS and to explore whether SIRT1 is a therapeutic target for ALS and the role of resveratrol in ALS.
Methods: SOD1-G93A transgenic mice were selected as the experimental group. According to the course of disease, they were divided into pre symptom (60 days) group, early symptom (Onset) group and end stage (End) group, and 90 days of wild type mice were selected as control group, 3 rats in each group, resveratrol (Sigma, 101237259) 0.375g, soluble in 7.5ml anhydrous ethanol, plus physiological saline to 50ml, and 7.5mg/ml At the same time, 15% ethanol physiological saline solution was used as solvent; 3 groups were treated, and 70dSOD1-G93A transgenic mice were selected and divided into blank group, solvent group and resveratrol group. Each group was divided into 2 subgroups according to the time of administration, and each group was given the resveratrol solution 30mg/kg/d intragastric perfusion (resveratrol (resveratrol) (resveratrol) (resveratrol) from 70D (pre symptom). In the resveratol group), the same volume of solvent in the stomach perfusion (the solvent group), without any treatment (blank group), continuous perfusion to the early symptom (20-30 days of continuous administration), end stage (50-60 days of continuous administration), after the injection of 10% chloral chloral (350mg/Kg weight) abdominal cavity injection anesthesia, killed mice, and the motor cortex and lumbar pulp quickly put into liquid nitrogen cold. Frozen, then stored at -80 centigrade refrigerator; 4% polyoxymethylene was perfused by heart, then peeled off the motor cortex and lumbar spinal cord, soaked with 4% polyformaldehyde or fixed the tissue with 2.5% glutaraldehyde, and observed the dynamic expression of SIRT1 in the motor cortex and lumbar spinal cord of SOD1-G93A transgenic mice by using Western blot technique and immunohistochemical technique. The changes of SIRT1 in motor cortex and lumbar spinal cord of SOD1-G93A transgenic mice after resveratrol were analyzed, and data were analyzed by SPSS13.0.
Results: the dynamic changes of SIRT1 in the motor cortex and lumbar spinal cord of 1SOD1-G93A transgenic mice: the expression of SIRT1 in the motor cortex and lumbar spinal cord of SOD1-G93A transgenic mice showed that the expression of SIRT1 in the 60d group had no significant changes compared with the control group, and the SIRT1 expression in the early and end stage group of the 60d group increased. Histochemical staining showed that the expression of SIRT1 increased in the early stage of symptoms, and the motor neurons in the lumbar spinal cord decreased significantly in the end stage.
2 the changes in the expression of SIRT1 in the motor cortex and lumbar spinal cord of SOD1-G93A transgenic mice after resveratrol: after Wstern blot showed resveratrol, the expression of SIRT1 in the early motor cortex and the resveratrol group in the lumbar spinal cord was lower than that in the solvent group, and the expression of SIRT1 in the early solvent group was more than that in the blank group. There was no obvious change in the expression of SIRT1 between the end stage groups. Immunohistochemical staining showed that the expression of SIRT1 in the motor cortex of SOD1-G93A transgenic mice and the resveratrol group in the lumbar spinal cord was less than that in the solvent group, and the expression of SIRT1 in the resveratrol group was higher than that in the blank group, but there was no obvious change in the group of resveratrol group, but the SIRT1 expression in the solvent group was more obvious than that in the blank group. There was no significant difference in the expression of SIRT1 between the three groups in the end stage.
Conclusion: with the progress of the disease, the expression of SIRT1 in the motor cortex and lumbar spinal cord of SOD1-G93A transgenic mice increased gradually, and the expression of SIRT1 was less than that in the blank group when the resveratrol was given early in the symptom, and the expression of SIRT1 in the given solvent group was significantly higher than that of the resveratrol group and the blank group. There was no obvious change between the last groups, which said that veratrol did not activate SIRT1 and even played a role in reducing SRT1. Resveratrol did not have the expected protective effect on ALS. The protective effect of resveratrol was limited in the ALS model. The mechanism of resveratrol was still inconclusive. It needs further study and ALS The study plays a certain reference role.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R746.4

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