病毒硫氧还蛋白基因在Neuro2A细胞中的重组与表达

Thioredoxin gene recombination and expression in Neuro-2A cells

背景:目前对抗氧化基因硫氧还蛋白的研究逐步受到重视,但从基因治疗的角度对其研究较少。目的:采用硫氧还蛋白转染Neuro-2A细胞,观察细胞内表达相应蛋白因子对细胞的具体保护效果,并分析其发挥保护作用的可能机制。方法:以质粒PIRES2-EGFP-TRX转染Neuro-2A细胞,经RT-PCR鉴定,建立能够稳定表达硫氧还蛋白的细胞株。利用不同浓度的H2O2处理正常细胞及转染细胞,建立氧化应激模型。观察受到氧化损伤后两组细胞的形态学、存活率、还原型谷胱甘肽的浓度及细胞内DNA链断裂情况。结果与结论:经H2O2作用后,两组细胞形态均出现损伤,但转染组细胞比正常组细胞损伤减轻、细胞存活率升高、细胞内还原型谷胱甘肽水平增高、DNA链断裂程度减轻。说明人类硫氧还蛋白基因可以在Neuro-2A细胞中被重组并顺利表达,对细胞具有一定的保护作用;这一作用可能是通过清除氧自由基,维持细胞内还原型谷胱甘肽水平,从而保护细胞DNA免受氧化性损伤来实现的。

BACKGROUND： Studies on antioxidant genes Thioredoxin （TRX） have attracted more attention gradually but relevant studies from the aspect of gene therapy are fewer. OBJECTIVE： To study the protective effect of cells expressing the corresponding proteins in TRX transfected Neuro-2A cells and to analyze the possible mechanism underlying the protective effect METHODS： Plasmids PIRES2-EGFP-TRX were used to transfect Neuro-2A cells and to establish a cell line which could stably express TRX proteins identified by reverse transcription-PCR. Different concentrations of hydrogen peroxide were used to treat normal cells and transfected cells for the establishment of oxidative stress models. Cell morphology, cell survival, glutathione concentration and DNA strand breaks situation were observed after oxidative damage. RESULTS AND CONCLUSION： Normal cells and transfected cells were both damaged by hydrogen peroxide; however, the transfected cells were superior to the normal cells in cell damage, cell survival, intracellular glutathione level and degree of DNA strand breaks. These findings indicate that TRX gene in Neuro-2A cells can be reconstructed and expressed successfully, which plays a certain protective. Thiseffect may be achieved through scavenging oxygen free radicals, maintaining intracellular glutathione levels thereby protecting cellular DNA against oxidative damage.