| Objective: By lentivirus-mediated Keap1 gene silencing of human keratinous form cell line HaCaT cells as the cell model, study the influence of Nrf2 on keratinizing related gene and arsenic metabolism related indicators in arsenic induced skin toxicity, and provide a theoretical basis for arsenic induced skin toxicity mechanism Methods: 1.shRNA Keap1 Construction:the use of RNAi technology, the human Keap1 gene as the target gene, design the specific small interfering RNA of Keap1 gene,then construct its shRNA, recombinant lentivirus expression of the sequencing of identification. 2.lentiviral packaging: using recombinant successful expression lentivirus was transfected into packaging cell line 293 T cell, collect and concentrated lentivirus supernatant,determination of the titer. 3.Keap1 silence HaCaT cell model construction: construction of three types of lentivirus were infected HaCaT cells(MOI=5), 48 hours after infection,1ug/ml of puromycin screening stable cell strains. 4.Stable cell lines identifying: the stable cell lines were screened, ues real-time Quantitative PCR to detected the interference efficiency. 5.Cell culture and exposure: model cells and normal cells by cell culture method. 6.MTT method to detect cell growth status, determine the concentration of exposure, the concentration of 1/100, 1/50, 1/10 of LC50. 7.Flow cytometry was used to detect the expression level of the apoptosis to the cells. 8.real time fluorescence quantitative PCR detection the mRNA expression of Nrf2, K1, K10, INV, LOR, NFkappa B, N6AMT1, in cells. 9.ELISA method was used to detect the expression level of SAM, HCY,N6AMT1, and COX-2 in cells. Results: 1.The results showed that the recombinant lentivirus was constructed successfully. 2.Lentivirus titer test results:according to the titer(TU/ml) = the number of cells×percentage×MOI(1)×virus dilution ratio of×10^3,to select the best titer of lentivirus. 3.To select Hacat Keap1shRNA1 cells,the gene silencing efficiency was 71%. 4.Mixed arsenic liquid respectively were 2.90μmol/L, 5.80 μmol/L, 29 μmol/L, low, medium and high dose group,used 0μmol/L to exposure control group and blank control group. 5.Cell proliferation and Cell apoptosis:2.90 μmol/L mixed fluid arsenic infected cells,the cells obviously proliferation and reduce apoptosis;With concentration of time,the cell proliferation rate drop,apoptosis,prompt mix of arsenic on time-measuring dependent cell proliferation and apoptosis. 6.2.90 μmol/L, 5.80 μmol/L mixed arsenic exposure Keap1 silenced cells at 8h, 24 h could promote the mRNA of Nrf2, K1, K10, INV, N6AMT1, NF-kappa B upregulation; 29.00μmol/L mixed arsenic exposure Keap1 silenced cells 72 h the mRNA of Nrf2, K1, K10,LNV, N6AMT1, NF-kappa B expression were down regulated,the difference was statistically significant(P<0.05). 7. 2.90 μmol/L, 5.80 μmol/L mixed arsenic exposure Keap1 silenced cells can promote the expression of N6AMT1, COX-2, SAM, HCY protein; 29.00 μmol/L mixed arsenic exposure Keap1 silenced cells the expression of N6AMT1, Sam, Hcy protein were decreased,the difference has statistical significance(P<0.05); 29.00 μmol/L of COX-2 protein expression was still decrease, greater than the control group and the difference is statistically significant(P<0.05). Conclusion: 1.The results of cell proliferation and apoptosis were described. 2.Mixed arsenic exposure Keap1 silencing HaCaT cells, Nrf2 was in a high expression state, with prolonged exposure time,increased concentration of the gene has a downward trend, but was significantly higher than the non exposure group. 3.Mixed arsenic exposure can make Keap1 silence HaCaT cells in K1, K10, INV, N6AMT1, NF-kappa B mRNA expression changes, with the exposure time prolonged, the concentration of exposure to increase can make the gene expression gradually down. 4.Keap1 silencing HaCaT cells after exposure in 8h, 24 h was not detected LOR mRNA expression; in the middle dose, low dose in 72 h, high dose group in 48 h,72h, detected the expression of LOR mRNA up regulation. 5.Mixed arsenic exposure can make Keap1 silence HaCaT cells the protein expression of N6AMT1,COX-2, SAM, HCY change, with the exposure time prolonged, increased exposure concentration can make the protein content decreased gradually. 6.Effects of toxicity of mixed arsenic on Keap1 silencing HaCaT cells is affected by time and interaction of concentration,and 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. |
PDF Full Text Request