本文選題：肌萎縮側(cè)索硬化 + 長壽蛋白��； 參考：《河北醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：肌萎縮側(cè)索硬化（Amyotrophic lateral sclerosis,ALS）是一種同時(shí)累及上、下運(yùn)動神經(jīng)元，及其支配的軀干、四肢和頭面部肌肉的慢性進(jìn)行性變性疾病。臨床上常表現(xiàn)為上、下運(yùn)動神經(jīng)元合并受損的混合性癱瘓。該病自發(fā)現(xiàn)以來已有100多年的歷史，雖經(jīng)大量研究，其發(fā)病機(jī)理仍不清楚，仍無有效的治療方法，病人的生活質(zhì)量差，平均存活期短，僅有3-5年。目前關(guān)于ALS發(fā)病機(jī)制的假說有很多，比如興奮性氨基酸毒性、氧化應(yīng)激、免疫學(xué)機(jī)制、線粒體功能障礙和異常蛋白的聚集等。然而，選擇性運(yùn)動神經(jīng)元丟失的根本原因依然不詳，目前仍無特效治療方法。 SIRT1（sirtuins）又名長壽蛋白，最早發(fā)現(xiàn)于酵母菌中，是一類從細(xì)菌到人類都高度保守的NAD+依賴的去乙�；鞍酌割悾制邆�(gè)亞型，分別為SIRT1-SIRT7,研究最多的是SIRT1，它與酵母菌的SIR2基因的同源性最高，可以使組蛋白去乙�；{(diào)節(jié)許多蛋白酶類的活性，還能從基因水平影響蛋白的表達(dá)，參與脂肪代謝、能量限制、免疫功能、炎癥反應(yīng)、自噬、增殖與老化、細(xì)胞凋亡、腫瘤等生理功能。近年來，人們對sirtuins在2型糖尿病、癌癥、心血管疾病等多種疾病和其相關(guān)并發(fā)癥、遺傳性疾病和神經(jīng)退行性變疾病中的研究日益增多。作為一種保護(hù)性蛋白，在朊蛋白病模型的研究中，SIRT1可以通過保護(hù)線粒體來維持細(xì)胞生存；帕金森病（PD）模型中，SIRT1可以對抗氧化應(yīng)激損傷；阿爾茨海默�。ˋD）中，有研究指出SIRT1可以減少β淀粉樣蛋白前體（APP）形成，減少β淀粉樣蛋白沉積。 白藜蘆醇（Resveratrol，Res）是多酚類化合物，已被證實(shí)可以激活SIRT1并且廣泛用于各種實(shí)驗(yàn)中，是近來研究較多的SIRT1的激動劑，它主要來源于蓼科植物的根莖中。白藜蘆醇具有抗炎、抗癌、抗氧化、抗心血管疾病等作用，近來對于白藜蘆醇在神經(jīng)退行性疾病中有益作用的研究日益增多。 然而，在同樣作為神經(jīng)變性疾病的ALS中，SIRT1的表達(dá)有無變化，有學(xué)者證實(shí)SIRT1在ALS中表達(dá)是增多的，但也有學(xué)者證實(shí)SIRT1是減少的，目前其在ALS中的作用及表達(dá)變化還未有定論，我們所感興趣和要研究的是隨著疾病的進(jìn)展，SIRT1在ALS小鼠運(yùn)動皮層與腰髓中會有怎樣的動態(tài)變化以及給予白藜蘆醇后對SIRT1的表達(dá)量會產(chǎn)生怎樣的影響。 SOD1-G93A轉(zhuǎn)基因小鼠是目前研究ALS較為理想的動物模型之一，本實(shí)驗(yàn)旨在觀察隨著疾病的進(jìn)展，SOD1-G93A轉(zhuǎn)基因小鼠運(yùn)動皮層及腰髓中SIRT1的動態(tài)變化，同時(shí)給予SIRT1激活劑白藜蘆醇后SOD1-G93A轉(zhuǎn)基因小鼠運(yùn)動皮層與腰髓中SIRT1的變化，來進(jìn)一步加深對ALS的研究，探討SIRT1成為ALS的治療靶點(diǎn)是否具有可行性和白藜蘆醇在ALS中的作用。 方法：選取SOD1-G93A轉(zhuǎn)基因小鼠為實(shí)驗(yàn)組，根據(jù)病程分為癥狀前期（60天）組、癥狀早期（Onset）組、終末期(End)組，選取90天的野生型小鼠作為對照組，各組3只；白藜蘆醇（Sigma，101237259）0.375g，溶于7.5ml無水乙醇中，加生理鹽水定容至50ml,配成7.5mg/ml的溶液，同時(shí)配制15%的乙醇生理鹽水溶液作為溶劑；處理組分3組，選取70dSOD1-G93A轉(zhuǎn)基因小鼠，分為空白組、溶劑組、白藜蘆醇組，，每組根據(jù)給藥時(shí)間分2個(gè)亞組，每組6只，從70d（癥狀前期）開始分別給予上述白藜蘆醇溶液30mg/kg/d胃內(nèi)灌注（白藜蘆醇組）、同等體積的溶劑胃內(nèi)灌注（溶劑組）、不給予任何處理（空白組），分別連續(xù)灌注至癥狀早期（連續(xù)給藥20-30天）、終末期（連續(xù)給藥50-60天）后取材，以10%水合氯醛(350mg/Kg體重)腹腔注射麻醉，處死小鼠，取運(yùn)動皮層與腰髓迅速投入液氮冷凍，之后保存于-80℃冰箱；4%多聚甲醛經(jīng)心臟灌注，之后剝離小鼠運(yùn)動皮層與腰髓，用4%多聚甲醛浸泡或用2.5%戊二醛固定組織，利用Western blot技術(shù)和免疫組織化學(xué)技術(shù)觀察SOD1-G93A轉(zhuǎn)基因小鼠運(yùn)動皮層與腰髓中SIRT1的動態(tài)表達(dá)情況及給予白藜蘆醇后SOD1-G93A轉(zhuǎn)基因小鼠運(yùn)動皮層及腰髓中SIRT1的變化。并利用spss13.0對數(shù)據(jù)進(jìn)行分析。 結(jié)果：1SOD1-G93A轉(zhuǎn)基因小鼠運(yùn)動皮層與腰髓中隨疾病發(fā)展SIRT1的動態(tài)變化：Wstern blot技術(shù)檢測SOD1-G93A轉(zhuǎn)基因小鼠運(yùn)動皮層與腰髓SIRT1表達(dá)結(jié)果顯示，與對照組相比，60d組SIRT1表達(dá)量無明顯變化，癥狀早期及終末期組SIRT1表達(dá)量均增加。免疫組織化學(xué)染色可見癥狀早期組、終末期組SIRT1表達(dá)增加，終末期腰髓中運(yùn)動神經(jīng)元明顯減少。 2給予白藜蘆醇后SOD1-G93A轉(zhuǎn)基因小鼠運(yùn)動皮層與腰髓中SIRT1的表達(dá)量變化：Wstern blot示給予白藜蘆醇后，癥狀早期運(yùn)動皮層及腰髓中白藜蘆醇組SIRT1表達(dá)量較溶劑組降低，較空白組無明顯變化；癥狀早期溶劑組SIRT1表達(dá)量較空白組增多。終末期各組之間SIRT1表達(dá)量均無明顯變化。免疫組織化學(xué)染色顯示癥狀早期SOD1-G93A轉(zhuǎn)基因小鼠運(yùn)動皮層與腰髓中白藜蘆醇組較溶劑組SIRT1表達(dá)減少；白藜蘆醇組SIRT1表達(dá)較空白組有所增加，但無明顯變化；溶劑組較空白組SIRT1表達(dá)明顯增加。終末期三組間SIRT1表達(dá)無明顯差異。 結(jié)論：隨著疾病的進(jìn)展，SOD1-G93A轉(zhuǎn)基因小鼠運(yùn)動皮層與腰髓中SIRT1的表達(dá)量逐漸增加，而當(dāng)癥狀早期給予白藜蘆醇后，SIRT1的表達(dá)量較空白組無明顯變化，較溶劑組減少，而給予溶劑組SIRT1表達(dá)量較白藜蘆醇組及空白組均有明顯增高，終末期各組之間均無明顯變化，這說明白藜蘆醇沒有起到激活SIRT1的作用，甚至起到了降低SRT1的作用，白藜蘆醇對于ALS未起到預(yù)期的保護(hù)效應(yīng)，其在ALS模型中的保護(hù)作用有限，白藜蘆醇的作用機(jī)制尚不能定論，需進(jìn)一步深入研究，同時(shí)對ALS的研究起到一定參考作用。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R746.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王穎;高靜;錢進(jìn)軍;;SIRT1與神經(jīng)變性疾病[J];生命科學(xué);2010年04期

本文編號：1931828