TRX基因在Neuro-2A细胞中的表达及其对氧自由基清除作用的研究

发布时间：2018-05-27 18:16

本文选题：TRX基因 + 转染　；参考：《大连医科大学》2012年硕士论文

【摘要】：目的：硫氧还蛋白（Thioredoxin,TRX）是广泛存在于真核生物神经细胞内的一种具有抗氧化应激功能的小分子蛋白。当细胞受到氧化损伤时，能在一定程度上清除氧自由基，从而保护细胞免受诸如肿瘤坏死因子和氢过氧化物的的损害。TRX对于机体神经细胞具有保护作用已经被证实，并且已逐渐引起人们对其具体保护效果的关注。一些研究显示，应用TRX处理后可使培养的神经元细胞存活率大幅度提高。虽然细胞实验显示TRX具有抗氧化功能，但对于TRX的具体保护效果的研究资料有限，以至迄今尚未把TRX作为一种外源性的治疗药物或治疗手段来研究并做出明确的结论。基因治疗是近年来分子生物学领域逐渐兴起的一种新的疾病治疗方法，可以将某种特定基因导入体细胞表达以产生特定的蛋白质因子，实现对疾病的治疗作用。结合本实验目的，我们把正常的人类TRX基因导入所要研究的异种生物细胞内，以观察TRX基因在细胞内的表达效果和保护作用，目的是研究TRX通过基因治疗途径来发挥其防治效果的可行性，并初步探讨TRX发挥抗氧化作用的可能机制。由于机体的中枢神经系统中含有大量的不饱和脂肪酸，故更易受到自由基的攻击，导致脂质过氧化等损伤，因此本研究选用小鼠神经嵴母细胞瘤（Neuro-2A）作为实验对象。Neuro-2A细胞是一种具有正常神经细胞形态和生理生化特性的神经元肿瘤细胞，保留了一系列正常神经细胞的功能，对于神经系统的实验研究具有较广泛的代表性。本实验旨在探讨TRX转染Neuro-2A细胞后，在细胞内表达相应蛋白因子，对细胞的具体保护效果，进一步补充TRX对神经系统具体保护效果的研究资料，进而为基因治疗的基础研究提供初步的方法学依据，也为临床治疗神经系统疾病的生物学治疗方法探索一条新的途径。方法：以Neuro-2A细胞作为实验对象，应用分子生物学技术，将质粒PIRES2-EGFP-TRX转染Neuro-2A细胞，挑出转染成功的细胞进行扩大培养，使其形成一个细胞株，并通过RT-PCR法检测细胞中TRX的mRNA的表达水平。将H2O2作用于Neuro-2A细胞，建立氧化损伤模型，观察转染组及非转染组细胞的外观形态和存活率。以四唑盐（MTT）法检测两组细胞的存活率，以苯二醛（OPT）测定两组细胞内还原型谷胱甘肽（GSH）水平，采用单细胞凝胶电泳（SCGE）实验检测两组细胞的DNA链断裂情况。实验结果用SPSS v11.5统计软件包进行统计分析。结果：一、转染后的Neuro-2A细胞于倒置荧光显微镜下观察，可见转染成功的细胞发出明亮的绿色荧光。应用RT-PCR法检测转染细胞中TRX的mRNA的表达水平，于分子量约318bp处可见清晰条带，而非转染细胞则未见条带。二、MTT法检测细胞存活率：用0.0625mM—2.0mM的H2O2作用于两组细胞3h后，两组细胞的存活率呈逐渐降低趋势；但在各损伤浓度中，转染组比非转染组细胞存活率增高（P0.05或P0.01）。三、细胞内GSH水平测定：用0.03125mM—0.5mM的H2O2作用于两组细胞2h后，引起细胞的GSH水平降低；但在各损伤浓度中，转染组比非转染组细胞内GSH水平增高（P0.05或P0.01）。四、彗星实验：用0.03125mM—0.125mM的H2O2作用于两组细胞1h后，引起细胞DNA链断裂，形成彗星样拖尾；但未转染组各损伤浓度的尾长、尾距及尾DNA含量均明显大于转染组（P0.05或P0.01）。结论：一、人类TRX基因可以在Neuro-2A细胞中被重组并顺利表达。二、TRX基因表达的TRX具有抗氧化应激的作用，对Neuro-2A细胞受到的氧化性损伤具有一定的保护作用，提高细胞的存活率。三、TRX对Neuro-2A细胞的保护作用可能是通过清除氧自由基，维持细胞内GSH水平，从而保护细胞DNA免受氧化性损伤来实现的。
[Abstract]:Objective: Thioredoxin (TRX) is a small molecular protein, which is widely distributed in eukaryotic cells. When cells are damaged by oxidative damage, they can deoxidize free radicals to a certain extent and protect cells from the damage of.TRX, such as TNF and hydroperoxide. The protective effect of nerve cells in the body has been confirmed and has gradually attracted attention to its specific protective effects. Some studies have shown that the use of TRX can greatly improve the survival rate of cultured neuron cells. Although cell experiments show that TRX has antioxidant function, the specific protective effect of TRX is studied. The data are limited, so far, TRX has not been studied and made clear conclusions as an exogenous therapeutic drug or therapeutic means.
Gene therapy is a new method of treating disease in the field of molecular biology in recent years. It can express certain genes into body cells to produce specific protein factors to achieve the therapeutic effect on disease. Combined with the purpose of this experiment, we import normal human TRX gene into the xenogenic cells to be studied. In order to observe the expression effect and protective effect of TRX gene in cells, the purpose of this study is to study the feasibility of TRX to play its control effect through gene therapy, and to explore the possible mechanism of TRX to play an antioxidant role.
Because there are plenty of unsaturated fatty acids in the central nervous system of the body, it is easier to be attacked by free radicals and lead to lipid peroxidation. Therefore, the mouse neural crest blastoma (Neuro-2A) is selected as the experimental.Neuro-2A cell as a neuron with normal neural cell morphology and physiological and biochemical characteristics. Tumor cells retain a series of functions of normal nerve cells, which are widely representative for the experimental study of nervous system.
The purpose of this study is to investigate the expression of the corresponding protein factors in the cells after transfection of TRX to Neuro-2A cells, the specific protective effect on the cells, and further supplement the research data on the specific protective effect of TRX on the nervous system, and then provide a preliminary methodology for the basic research of gene therapy, and also for the clinical treatment of the biological diseases of the nervous system. A new approach is explored for the treatment.
Methods: Neuro-2A cells were used as the experimental object. The plasmid PIRES2-EGFP-TRX was transfected into Neuro-2A cells by molecular biology technology, and the transfected cells were selected to expand culture to form a cell line, and the expression level of TRX mRNA in the cells was detected by RT-PCR. H2O2 was used as a Neuro-2A cell to establish oxidative damage. The appearance and survival rate of the cells in the transfected group and non transfected group were observed. The survival rate of the two groups of cells was detected by four azoles (MTT) method, and the level of the reduced glutathione (GSH) in the two groups was measured with benzene two aldehyde (OPT), and the DNA strand breaks of the two groups were detected by the single cell gel electrophoresis (SCGE). The experimental results were used in SPSS v11.. 5 statistical software package is used for statistical analysis.
The results were as follows: 1. The transfected Neuro-2A cells were observed under the inverted fluorescence microscope, and the successful transfected cells showed a bright green fluorescence. The expression level of TRX mRNA in transfected cells was detected by RT-PCR method. The clear bands were visible at the molecular weight of about 318bp, but no bands were found in the non transfected cells. Two, MTT method was used to detect the cell survival rate. The survival rate of two groups of cells decreased gradually after the action of 0.0625mM - 2.0mM H2O2 on two groups of cell 3h, but the survival rate of the transfected group was higher than that of the non transfected group (P0.05 or P0.01). Three, the intracellular GSH level was determined: the GSH level of the cells was induced by 0.03125mM 0.5mM H2O2 in the two group of cell 2H. The level of GSH in the cells of the transfected group was higher than that in the non transfected group (P0.05 or P0.01). Four, four, comet experiment: after the H2O2 of 0.03125mM 0.125mM was used in the two group of cell 1H, the cell DNA chain was broken and the comet like trailing was formed, but the tail length of the damage concentration, the tail distance and the tail DNA content of all the untransfected groups were obviously greater than those of the untransfected group. Transfection group (P0.05 or P0.01).
Conclusion: 1. The human TRX gene can be reorganized and expressed in Neuro-2A cells. Two, the TRX expressed by TRX gene has the effect of antioxidant stress, which can protect the oxidative damage of Neuro-2A cells and increase the survival rate of the cells. Three, the protective effect of TRX on Neuro-2A cells may be by scavenging oxygen free freedom. It is necessary to maintain GSH level in cells and protect DNA cells from oxidative damage.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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