發(fā)布時(shí)間：2018-05-27 18:16

  本文選題：TRX基因 + 轉(zhuǎn)染　； 參考：《大連醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的：硫氧還蛋白（Thioredoxin,TRX）是廣泛存在于真核生物神經(jīng)細(xì)胞內(nèi)的一種具有抗氧化應(yīng)激功能的小分子蛋白。當(dāng)細(xì)胞受到氧化損傷時(shí)，能在一定程度上清除氧自由基，從而保護(hù)細(xì)胞免受諸如腫瘤壞死因子和氫過(guò)氧化物的的損害。TRX對(duì)于機(jī)體神經(jīng)細(xì)胞具有保護(hù)作用已經(jīng)被證實(shí)，并且已逐漸引起人們對(duì)其具體保護(hù)效果的關(guān)注。一些研究顯示，應(yīng)用TRX處理后可使培養(yǎng)的神經(jīng)元細(xì)胞存活率大幅度提高。雖然細(xì)胞實(shí)驗(yàn)顯示TRX具有抗氧化功能，但對(duì)于TRX的具體保護(hù)效果的研究資料有限，以至迄今尚未把TRX作為一種外源性的治療藥物或治療手段來(lái)研究并做出明確的結(jié)論。 基因治療是近年來(lái)分子生物學(xué)領(lǐng)域逐漸興起的一種新的疾病治療方法，可以將某種特定基因?qū)塍w細(xì)胞表達(dá)以產(chǎn)生特定的蛋白質(zhì)因子，實(shí)現(xiàn)對(duì)疾病的治療作用。結(jié)合本實(shí)驗(yàn)?zāi)康�，我們把正常的人�?lèi)TRX基因?qū)胨芯康漠惙N生物細(xì)胞內(nèi)，以觀察TRX基因在細(xì)胞內(nèi)的表達(dá)效果和保護(hù)作用，目的是研究TRX通過(guò)基因治療途徑來(lái)發(fā)揮其防治效果的可行性，并初步探討TRX發(fā)揮抗氧化作用的可能機(jī)制。 由于機(jī)體的中樞神經(jīng)系統(tǒng)中含有大量的不飽和脂肪酸，故更易受到自由基的攻擊，導(dǎo)致脂質(zhì)過(guò)氧化等損傷，因此本研究選用小鼠神經(jīng)嵴母細(xì)胞瘤（Neuro-2A）作為實(shí)驗(yàn)對(duì)象。Neuro-2A細(xì)胞是一種具有正常神經(jīng)細(xì)胞形態(tài)和生理生化特性的神經(jīng)元腫瘤細(xì)胞，保留了一系列正常神經(jīng)細(xì)胞的功能，對(duì)于神經(jīng)系統(tǒng)的實(shí)驗(yàn)研究具有較廣泛的代表性。 本實(shí)驗(yàn)旨在探討TRX轉(zhuǎn)染Neuro-2A細(xì)胞后，在細(xì)胞內(nèi)表達(dá)相應(yīng)蛋白因子，對(duì)細(xì)胞的具體保護(hù)效果，進(jìn)一步補(bǔ)充TRX對(duì)神經(jīng)系統(tǒng)具體保護(hù)效果的研究資料，進(jìn)而為基因治療的基礎(chǔ)研究提供初步的方法學(xué)依據(jù)，也為臨床治療神經(jīng)系統(tǒng)疾病的生物學(xué)治療方法探索一條新的途徑。 方法：以Neuro-2A細(xì)胞作為實(shí)驗(yàn)對(duì)象，應(yīng)用分子生物學(xué)技術(shù)，將質(zhì)粒PIRES2-EGFP-TRX轉(zhuǎn)染Neuro-2A細(xì)胞，挑出轉(zhuǎn)染成功的細(xì)胞進(jìn)行擴(kuò)大培養(yǎng)，使其形成一個(gè)細(xì)胞株，并通過(guò)RT-PCR法檢測(cè)細(xì)胞中TRX的mRNA的表達(dá)水平。將H2O2作用于Neuro-2A細(xì)胞，建立氧化損傷模型，觀察轉(zhuǎn)染組及非轉(zhuǎn)染組細(xì)胞的外觀形態(tài)和存活率。以四唑鹽（MTT）法檢測(cè)兩組細(xì)胞的存活率，以苯二醛（OPT）測(cè)定兩組細(xì)胞內(nèi)還原型谷胱甘肽（GSH）水平，采用單細(xì)胞凝膠電泳（SCGE）實(shí)驗(yàn)檢測(cè)兩組細(xì)胞的DNA鏈斷裂情況。實(shí)驗(yàn)結(jié)果用SPSS v11.5統(tǒng)計(jì)軟件包進(jìn)行統(tǒng)計(jì)分析。 結(jié)果：一、轉(zhuǎn)染后的Neuro-2A細(xì)胞于倒置熒光顯微鏡下觀察，可見(jiàn)轉(zhuǎn)染成功的細(xì)胞發(fā)出明亮的綠色熒光。應(yīng)用RT-PCR法檢測(cè)轉(zhuǎn)染細(xì)胞中TRX的mRNA的表達(dá)水平，于分子量約318bp處可見(jiàn)清晰條帶，而非轉(zhuǎn)染細(xì)胞則未見(jiàn)條帶。二、MTT法檢測(cè)細(xì)胞存活率：用0.0625mM—2.0mM的H2O2作用于兩組細(xì)胞3h后，兩組細(xì)胞的存活率呈逐漸降低趨勢(shì)；但在各損傷濃度中，轉(zhuǎn)染組比非轉(zhuǎn)染組細(xì)胞存活率增高（P0.05或P0.01）。三、細(xì)胞內(nèi)GSH水平測(cè)定：用0.03125mM—0.5mM的H2O2作用于兩組細(xì)胞2h后，引起細(xì)胞的GSH水平降低；但在各損傷濃度中，轉(zhuǎn)染組比非轉(zhuǎn)染組細(xì)胞內(nèi)GSH水平增高（P0.05或P0.01）。四、彗星實(shí)驗(yàn)：用0.03125mM—0.125mM的H2O2作用于兩組細(xì)胞1h后，引起細(xì)胞DNA鏈斷裂，形成彗星樣拖尾；但未轉(zhuǎn)染組各損傷濃度的尾長(zhǎng)、尾距及尾DNA含量均明顯大于轉(zhuǎn)染組（P0.05或P0.01）。 結(jié)論：一、人類(lèi)TRX基因可以在Neuro-2A細(xì)胞中被重組并順利表達(dá)。二、TRX基因表達(dá)的TRX具有抗氧化應(yīng)激的作用，對(duì)Neuro-2A細(xì)胞受到的氧化性損傷具有一定的保護(hù)作用，提高細(xì)胞的存活率。三、TRX對(duì)Neuro-2A細(xì)胞的保護(hù)作用可能是通過(guò)清除氧自由基，維持細(xì)胞內(nèi)GSH水平，從而保護(hù)細(xì)胞DNA免受氧化性損傷來(lái)實(shí)現(xiàn)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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R363

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