Study On The Effect Of NRF-1 Gene Overexpression On Hypoxia Injury Of Rat Cardiomyocytes H9c2

Objcective: The model of hypoxia myocardial cell was established, which aimed at study the pathogenesis of heart failure and investigation whether overexpression of the NRF-1 gene could ameliorate the pathological injury caused by myocardial anoxia, meanwhile, laid foundations for search a new target gene to cure heart failure.Method:1. lentiviral vector was used to construct cell lines which high expressed NRF-1 gene and empty virus control cell lines.2. The two cell lines were used to construct hypoxia model induced by cobalt chloride, and the state energy level changes of the two cell lines were determined through the method of CCk-8 in hypoxia.3. Test the change of indexes on the cell hypoxia conditions.(1) Using specificity fluorescent dye of mitochondrial JC-1 and flow cytometry determined the change of mitochondrial membrane potential after hypoxia treatment.(2) Using Hoechst 33342 method determined the apoptosis ratios of different group cells under hypoxia conditions.(3) Using fluorescence quantitative PCR detected in hypoxia or normal state the expression level of NRF-1 overexpression cells, the downstream related target genes(mt TFA, mt SSB, ATPδ, Cyt c), coactivation factor(NRF-2), hypoxia inducing factor(HIF-1α) and apoptosis correlation factor(Bax, Bcl-2, Caspase3).Result:1. The expression level of NRF-1 gene in overexpression cell lines was higher than empty viruse group, and though antibiotics screening, we obtained stable transfection cell lines(NRF-H9c2 and p Lenti-H9c2).2. Successfully established the cell hypoxia model induced by cobalt chloride, and the level of cell-vitality was significantly reduced after induced by cobalt chloride.3. Under normal conditions, overexpression cells and virus control cells were equal on the level of mitochondrial depolarization, 2.54% and 2.60% respectively; When treated with 200μM cobalt chloride for 24 hours, cell depolarization levels turned into 6.72% and 9.6%; When the concentration increased to 400μM, the cells depolarization levels increased to 9.34% and 16.08% respectively; indicated that overexpression NRF-1 gene could reduce mitochondria damage that caused by cobalt chloride.4. The cell apoptosis rate of empty virus group was(15.9±3.4) % after cobalt chloride hypoxia treatment, there had statistically significant( p<0.01) compared with undealt. But in the group of overexpression NRF-1 gene, the cell apoptosis rate was(8.1±0.3)%, the apoptosis rate decreased compared with Empty virus treatment group and the result was statistically significant(p<0.01).5. Overexpression NRF-1 gene increased expression levels of downstream target genes(mt TFA gene and mt SSB gene), meanwhile, increased the level of coactivation NRF-2 gene of transcription factor, but it did not increase the express level of Cyt c gene and ATPδ gene. In addition, NRF-1 gene overexpression could increase the antiapoptotic factor(Bcl-2) expression and inhibit Bax expression; it also can reduce Caspase3 expression.Conclusion:1. In hypoxia condition, overexpression of the NRF-1 gene is beneficial to cell survival.2. NRF-1 gene improved cell hypoxia state, which might be through improving cell vitality, decreasing the level of cell mitochondrial depolarization, Increasing expression level antiapoptosis genes, and inhibiting gene expression of apoptosis genes to reduce the rate of apoptosis.3.The preliminary confirmation was that NRF-1 gene was expected as a candidate molecules of gene therapy for heart failure...