The Influence Of Expression Of Gene Related Nrf2 And Role In The Skin Oxidative Stress Injury Caused By Arsenic Through Silencing Keap1 Gene

Objective:Investigating the effects of erpression of gene related Nrf2 and role in the skin toxicity cansed by arsenic through silencing Keap1 gene for further elaboration of arsenic inducing skin injury in early stage.Methods:1.ShRNA isotopic carrier was constructed. The designed of shRNA are according to the gene sequence of Keap1.Using BamH I and EcoR I to restrict isotopic carrier,T4 ligase to connect isotopic carrier and target gene.Picking the culture germs and sequencing,after competent cells was transformed.2.Ientiviral packaging.Extracting the positive plasmid,and then transfection293 T cells, collect and concentrated lentivirus supernatant,determanation of the titer.3.The mode of cells of silencing Keap1 was constructed. Three types virus was constructed to infect HaCaT cells(MOI=5).Using 1ug/ml of puromycin to screen stable cell strains after48 hours.4.Identifyings of table cell lines.Cultivating the stable cell,then using real-time Quantitative PCR to detect the interference efficiency.5.MTT method to detect cell growth status,determine the concentration of exposure,the concentration of 1/100, 1/50, 1/10 of LC50.6.Flow cytometry was used to detect the expression level of the apoptosis to the cells.7.Real time fluorescence quantitative PCR detection the mRNA expression of Nrf2,Keap1,Bach1,ERK,CBP,AS3 MT,in cells.8.ELISA method was used to detect the expression level of AS3 MT,GSH, and HO-1 in cells.Results:1.The results showed that the recombinant lentivirus was constructed successfully.2.Virus titer test results:virus titer was2×108TU/ml.3.To select HaCaT KEAP1shRNA1 cells,the gene silencing efficiency was71%.4.Mixed arsenic liquid respectively were 2.90、5.80、29μmol/L,low,medium and high dose group,used 0μmol/L to exposure control group and blank control group.5.2.90μmol/L mixed arsenic could inhibit the apoptosis and promote the proliferation of HaCaT cells,with the prolonging of time and dose the apoptosis of cells were increased and the proliferation of cells were decreased.6.2.90、5.80μmol/L mixed arsenic by acting on the Keap1 silenced cells at 8h, 24 h could promote the expression of Nrf2,Keap1,Bach1, ERK,CBP,AS3 MT mRNA;29.00μmol/L mixed arsenic by acting on the Keap1 silenced cells72 h could refrain the expression of Nrf2,Keap1,Bach1,ERK,CBP,AS3 MT mRNA, the difference was statistically significant(P<0.05).7.2.90 、 5.80μmol/L mixed arsenic by acting on the Keap1 silenced cells at 8h,24 h could promote the expression of As3MT、GSH and HO-1 protein;29.00μmol/L mixed arsenic by acting on the Keap1 silenced cells at 72 h could refrain the expression of As3MT、GSH and HO-1 protein,the difference has statistical significance(P<0.05). Conclusion:1.Mixed arsenic exposure made mode cells change the rate of proliferation and apoptosis,low dose could promote the proliferation of cells and high dose promote the apoptosis of cells.2.Mode HaCaT cells was exposed in mixed arsenic,Nrf2 was in a high expression state,and with the prolonging of the exposure time and the increased concentration the gene has a downward trend,but was significantly higher than the non exposure group.3.Mixed arsenic exposure made mode cells change the expression of in Nrf2,Keap1,Bach1,ERK,CBP,AS3 MT mRNA and As3 MT 、 GSH and HO-1 protein expression,with the exposure time prolonged, the concentration of exposure to increase made the gene expression gradually down.4.Effects of toxicity of mixed arsenic on mode cells is affected by time and concentration,it may be speculated that arsenic exposure can make the damage to skin cells more serious with the extension of exposure time and the increase of exposure concentration.