The Effects Of Genes And Proteins Related Of Skin Keratinization Of Inhibiting Nrf2 Gene In HaCaT Cells Treated By Arsenic

Objective:INVestigating the effects of erpression of gene related Nrf2 and role in the skin toxicity cansed by arsenic through inhibitin Nrf2 gene for further elaboration of arsenic inducing skin injury in early stage.Methods:1.Construction of lentiviral vector:Nrf2 shRNA was constructed according to Nrf2 gene sequence,recombinant lentiviral expre-ssion vector was sequenced and identified.2.Lentivirus packaging: a large number of positive plasmids were extracted and amplified,and 293 T cells were collected.3.Constru-ction of Ha CaT cell line Nrf2 gene suppression: different lentiviral vector(vector,Nrf2 shRNA1/2/3)were used to infect HaCaT cells,multiplicity of infection(MOI)was 5,48 hours later,with 1 g/ml puromycin stable strain.4.Identification of transgenic plants:the stable strain was screened and identified by RNA,and the expression of Nrf2 was detected by real-time fluorescence quantitative(Real-Time Quantitative PCR).5.MTT reduction method was used to detect the growth status of cells,and the concentration of LC50 was determined.The concentrations were 1/100,1/50,1/10,LC50,respectively.6.Cell apoptosis was detected by flow cytometry.7.RT-PCR was used to detect the expression levels of Nrf2,Keap1,NF-,B,CBP,As3 MT,K-1,K-10,LOR,and mRNA in the cells of INV.8.The expression levels of HO-1,SAM,HCY and As3 MT were detected by ELISA kit.Results:1.Lentiviral vector construction,gene sequencing,and the target gene sequence completely matched,slow virus titer of 2 * 108TU/ml.2.The Nrf2 gene of the stable cell line SH3 was the best,and its gene silencing efficiency was 87%.3.The LC50 of mixed arsenic exposure modell cell 48 h was 210 mol/L,and the concentration of1/100 was LC50(mol/)L,LC50(),1/50(mol/L),LC50(1/10 21 mol/L).4.Inhibition of Keap1 gene and 2.10 mol/L mixed arsenic exposure promotes cell proliferation,cell growth speed;with the increase of exposure concentration(4.20 mol/L-21.00 mol/L)exposure time(48h,72h)cells,multinucleated cells appear obvious,off the wall.5.Compared with the Nrf2 inhibited control group,8h and 24 h 2.1 mol/L,4.20 mol/L,21mol/L concentration group,the early apoptosis rate was increased with the exposure dose increased,the apoptosis rate increased after 48h;the early apoptosis rate were decreased,the differences were statistically significant(P<0.01).The early apoptosis rate of 72 h group was lower than that of control group,the difference was statistically significant(P<0.01).6.Compared with the Nrf2 inhibited control group and 8h control group,Keap1,48 h,24h inhibited 72hKeap1 mRNA expression was significantly decreased(P<0.01),and decreased with time;2.1 mol/L,4.2 mol/L,21 mol/L Keap1 mRNA concentration group the expression of 24 h and 48 h decreased(P<0.01),with the concentration of expression increase the upward trend.With Nrf2 inhibited compared with the control group,blank control group and Keap1 control group 72hNrf2 inhibited mRNA expression was raised(P<0.01);2.1 mol/L 24 h,48h and 72hNrf2 concentration of group mRNA were up-regulated(P<0.01);and 4.2 mol/L and 21 mol/L 24 h,72hNrf2 mRNA expression was significantly decreased(P<0.01).With the blank control group and Nrf2 inhibition compared with the control group,low concentration group(2.1,4.20 mol/L)up-regulated NF-kappa B,CBP expression of mRNA;the high concentration group(21mol/L)long time(48h,72h)expression of NF-kappa B,CBP mRNA,the differences were statistically significant(P< 0.01).The expression of K-l of each group was mRNA in24 h and 48 h when compared with the blank control group and Nrf2 control group 72 h inhibition reduced,significantly increased,the difference was statistically significant(P<0.01);and the blank control group and Nrf2 inhibition compared with the control group,low concentration group(2.10,4.20 mol/L)long time(48h,72h)increased the K-l0 expression of mRNA,high concentration group K-l0 total mRNA was down regulated,the differences were statistically significant(P<0.01).With the blank control group and Nrf2 inhibition compared with the control group,low concentration group(2.10,4.20 mol/L),INV LOR mRNA and 24 h 72h expression was significantly increased,the expression of48 h and 21 mol/L,LOR concentration of group INV after mRNA24 h was reduced,the differences were statistically significant(P<0.01).7.Compared with the blank control group and Nrf2 inhibition the control group,4.2 mol/L and 21 mol/L mixed arsenic exposure promotes As3 MT expression of mRNA,down 2.10,4.20 mol/L,24 h concentration of group 8h and 48hAs3 MT protein content,expression of 72 h,21 mol/L48 h 72h As3 MT,head of the concentration time on protein content,the differences were statistical significance(P<0.01).8.Compared with the control group,Keap1 control group,48 h and 8h inhibited 72 hSAM protein content decreased(P<0.01);Nrf2 control group,48 h and 24 h inhibited 72 hSAM protein content was increased(P<0.01);and Nrf2 inhibition compared with the control group,2.1 mol/L,4.2 mol/L,21 mol/L concentration group with time(24h,48 h,72h)SAM(P<0.01)protein content declined at the same time,the different concentration was decreased as the concentration increased,the differences were statistically significant(P<0.01).With the blank control group and Nrf2 inhibition compared with the control group,8h,24 h,48h and 72 hHCY protein content was increased,the differences were statistically significant(P<0.01);the control group and Keap1 control group 24 h inhibited HCY protein content after inhibition of Nrf2 were lower than the control group,the difference was statistically significant(P<0.01).With the blank control group and Nrf2 inhibition compared with the control group,2.10,4.20 mol/L concentration group HO-1 protein content by 8h and 24 h to 48 h,the expression of 72 h expression decreased gradually 21 mol/L concentration group of HO-1 protein content to maintain the basic level down;at the same time different concentration groups with concentration increased the expression decreased gradually,the differences were statistically significant(P<0.01).Conclusion:1.Mixed arsenic exposure can change the ratio of cell proliferation and apoptosis,low dose promote cell proliferation,inhibit cell apoptosis,inhibit cell proliferation and promote cell apoptosis.2.Nrf2 pathway related genes(Nrf2,Keap1,NF-κB,CBP,As3MT)and proteins(HO-1,SAM,HCY,and so on)and the genes of Keratinized protein(K-1,K-10,INV,LOR)expression disorder were induced by arsenic-induced oxidative stress and Nrf2 gene inhibited.And they can promotes Oxidative stress response by Arsenic.3.With Nrf2 gene inhibited,Effects of toxicity of mixed arsenic on model cells is affected by time and concentration,it may be speculated that arsenic exposure can increased oxidative damage to skin cells more serious.