TRX Gene Expression And Clean Out Oxygen Free Radical Effect In Neuro2A Cells

Objective: TRX(Thioredoxin) is the small proteins have anti-oxidative stressfunction widely present in nerve cells. When the cells by oxidation injury, it canscavenging oxygen free radicals to a certain extent, thereby protecting cells from suchas tumor necrosis factor and hydrogen peroxide damage. TRX for the nerve cells have aprotective effect has been confirmed, and has gradually attracted people’s attention to itsspecific protective effect. Some studies show that application of TRX treatment incultured neurons can significantly improve cell survival. Although cell experimentsshowed that TRX has antioxidant function, but for the specific protective effect of TRXresearch data is limited,and even never take the TRX as an exogenous therapeutic drugsor treatments to study and make a clear conclusion.Gene therapy is the gradual emergence of a new disease treatments in the field ofmolecular biology in recent years, we can take a specific gene expression into somaticcells to produce specific protein factors to achieve the therapeutic effect of the disease.With the purpose of this study, we can take the normal human TRX gene into the studybiological cells in order to observe the TRX gene expression results in cells andprotection, purpose is to study TRX treatment feasibility through gene therapy, and toexplore the role of TRX may play an antioxidant mechanism.Because of the body’s central nervous system contains large amounts ofunsaturated fatty acids,so it is more vulnerable to free radical attack, resulting in lipidperoxidation and other damage, Therefore, this study used Neuro-2A as subjects.Neuro-2A is a nerve cell with normal morphology and physiological characteristics ofneuronal tumor cells,retaining a series of normal nerve cell function,for theexperimental study of the nervous system has a wider representation.This study was designed to explore the TRX transfected Neuro-2A cells,expressing the corresponding protein factor and the specific protective effect on cells, further supplement the TRX specific protective effect on the nervous system,andprovide a preliminary basis for gene therapy research methodology,and also for clinicaltreatment of neurological diseases to explore the biological treatment of a new way.Methods:To Neuro-2A cells as experimental subjects.Application of molecularbiology techniques, make the plasmid PIRES2-EGFP-TRX transfected Neuro-2Acells,singled out the transfected cells and extended training to form a cell line,and byRT-PCR assay in the mRNA expression levels of TRX.Make H2O2in Neuro-2Acells,establishment of oxidative damage model and observe transfected andnon-transfected cells in morphology and survival. We used the MTT assay measuringtwo sets cells livability,OPT measuring two sets cells GSH level, SCGE assay toelucidate the oxidative DNA damage.The data were statistically analyzed by SPSS v11.5software.Results:1. Transfected Neuro-2A cells were observed under an invertedfluorescence microscope, showing that cells transfected glows bright green fluorescence.By RT-PCR assay measuring TRX mRNA expression levels in transfected cells,and wecan seen it clear at the molecular weight of about318bp bands, non-transfected cells nobands.2. MTT assay measuring two sets cells livability:After0.0625mM-2.0mM ofH2O2treating for two groups of cells3h, the survival rate of two sets of cells wasgradually decreased; However, the damage in the transfected group than non-transfectedgroup increased cell survival rate(P<0.05or P<0.01).3.GSH assay: After0.03125mM—0.5mM of H2O2treating for two groups of cells2h,the level of GSH wasdecreased,we observed that transfected group increased the GSH level thannon-transfected group(P<0.05or P<0.01).4. SCGE assay: After0.03125mM—0.125mM of H2O2treating for two groups of cells1h,the DNA ruptureand form a comet-like tail,the damage in the transfected group less than thenon-transfected group(P<0.05or P<0.01).Conclusion:1. Human TRX gene can be recombined in Neuro-2A cells and wassuccessfully re-expression.2. TRX gene expression protein TRX exhibits protectionagainst oxidative stress,have a protective effect for nerve cells,and improve cell survival.3.The protective effect of TRX on the Neuro-2A cells may be through scavengingoxygen free radicals,maintenance of intracellular GSH levels,protecting Neuro-2A cellsfrom oxidative DNA damage to achieve.