阻滞NRF2后砷对NRF2抑制因子的影响及与MAPK基因的关系

发布时间：2018-03-06 04:19

本文选题：砷　切入点：HaCaT细胞　出处：《新疆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:NRF2基因抑制的人角质形成细胞株HaCa T细胞为模型细胞,阻滞NRF2对NRF2抑制因子的影响及与MAPK基因关系,为砷致皮肤毒性机制提供理论依据。方法:1、NRF2 shRNA的构建:以人源NRF2基因为靶基因,设计与NRF2基因具有特异性的小干扰RNA,构建其shRNA,重组慢病毒表达载体,并进行测序鉴定。2、慢病毒包装:用重组后表达成功的载体转染并包装细胞293T,收集、浓缩病毒上清液、测定其滴度。3、抑制NRF2基因的Hacat细胞模型的构建,构建好的三种慢病毒分别感染HaCaT细胞(MOI=5),感染48小时后,用1μg/ml的嘌呤霉素筛选稳定细胞株。4、稳定细胞株的鉴定:筛选出稳定的细胞株后,用实时荧光定量PCR(Real-Time Quantitative PCR)检测干扰效率。5、细胞培养与砷处理:所用细胞均按细胞常规培养方法进行培养。6、MTT法检测细胞生长状况,确定砷处理浓度,砷处理浓度别为以毒理学标准的LC50的1/100、1/50、1/10。7、流式细胞仪检测细胞活性氧。8、实时荧光定量PCR检测细胞中Nrf2、KEAP1、N6AMT1、Bach1、MAPK/ERK mRNA的表达水平。9、ELISA法检测细胞中GSH、N6AMT1、COX-2的蛋白表达水平。结果:1、慢病毒干扰载体构建测序结果显示重组载体构建成功。2、慢病毒滴度测试结果:依据滴度(TU/ml)=细胞数×百分比×MOI(1)×病毒稀释倍数×103选取最佳滴度的病毒。3、选取NRF2 shRNA3 Hacat细胞,其基因的抑制效率为87%。4、砷处理低、中、高剂量组分别为2.10μmol/L、4.20μmol/L、21.00μmol/L,抑制Nrf2、抑制Keap1、空白对照组砷处理浓度为0μmol/L。5、细胞增殖:2.10μmol/L砷处理细胞,细胞出现增殖现象;4.20μmol/L砷处理细胞在72h开始出抑制;21.00μmol/L砷处理细胞48h开始明显抑制细胞。空白组细胞增值率略低于抑制Keap1对照组,高于抑制Nrf2对照组。6、活性氧(ROS)在空白组表达较高随时间变化趋向稳定,抑制Keap1基因ROS表达上调,抑制Nrf2基因ROS降低,砷处理后ROS下调。7、2.10μmol/L、4.2μmol/L砷处理抑制Nrf2基因的细胞8h、24h后促进Bach1、N6AMT1、MAPK/ERK mRNA的表达;21.00μmol/L砷处理72h可使抑制Nrf2基因的细胞中Bach1、N6AMT1、MAPK/ERK mRNA表达下调,差异有统计学意义(P0.001)。8、2.10μmol/L、24h砷处理的抑制NRF2基因的HaCaT细胞能使N6AMT1、COX-2、GSH蛋白的表达上调;72h、29.00μmol/L砷处理抑制NRF2基因的HaCaT细胞使N6AMT1、COX-2、GSH蛋白的表达下调,差异有统计学意义(P0.05);结论:1、砷具有低促效应。2、N6AMT1能够调控氧化与抗氧化水平。3、GSH调节细胞氧化水平。4、ROS在体内有维持动态平衡。5、Bach1、COX-2能够参与抗氧化反应。6、MAPK/ERK会激活COX-2的表达。
[Abstract]:Objective to block the effect of NRF2 on NRF2 inhibitory factor and its relationship with MAPK gene in human keratinocyte line HaCa T cell line (HaCa T cell line), which is inhibited by 1: NRF2 gene. Methods: human NRF2 gene was used as target gene to design small interference RNAs specific to NRF2 gene and construct its shRNAs and recombinant lentivirus expression vector. DNA sequencing was performed to identify lentivirus packaging: the recombinant vector was successfully transfected and packaged into 293T, then the supernatant of virus was collected and concentrated, the titer of the supernatant was determined, and the Hacat cell model of inhibiting NRF2 gene was constructed. After 48 hours of infection, stable cell line. 4 was screened with 1 渭 g / ml purine mycin, and the stable cell line was identified. The interference efficiency. 5, cell culture and arsenic treatment were detected by real-time fluorescence quantitative PCR(Real-Time Quantitative. All the cells were cultured according to the conventional cell culture method. The growth status of the cells was detected by using the method of cell culture, and the concentration of arsenic treatment was determined. Arsenic concentration was measured as 1 / 100 / 1 / 50 / 10.7 of LC50 according to toxicological standard. Reactive oxygen species was detected by flow cytometry. Real time fluorescence quantitative PCR was used to detect the expression level of MAPKERK mRNA in cells. 9 Elisa was used to detect the expression of COX-2 protein in cells with GSHN6AMT1. The results showed that the expression level of COX-2 was detected by Elisa. The construction and sequencing of the virus interference vector showed that the recombinant vector was successfully constructed. The results of lentivirus titer test were as follows: according to the titer of NRF2 shRNA3 Hacat cells, the best titer of virus was selected according to the titer tu / ml = the number of cells 脳% 脳 moi 1) 脳 virus dilution times 脳 103, and NRF2 shRNA3 Hacat cells were selected. The inhibition efficiency of its gene was 87.4.The low arsenic treatment, the medium and high dose groups were 2.10 渭 mol / L ~ 4.20 渭 mol / L ~ 4.20 渭 mol 路L ~ (-1) 路L ~ (-1), respectively, which inhibited Nrf2 and Keap1. The arsenic concentration of control group was 0 渭 mol / L 路L ~ (5), and the proliferation of arsenic treated cells was 2.10 渭 mol / L ~ (-1). The proliferation of arsenic treated with 4.20 渭 mol/L began to inhibit 21.00 渭 mol/L arsenic treatment at 72 h, and the cell proliferation rate of the blank group was slightly lower than that of the control group, and the proliferation rate of the cells in the blank group was slightly lower than that in the control group. Compared with the control group, the expression of Ros in the blank group tended to be stable, the expression of Keap1 gene ROS was up-regulated, and the Nrf2 gene ROS was decreased, and the expression of Ros was higher than that of the control group (P < 0.05). After arsenic treatment, ROS down-regulated 2.10 渭 mol / L ~ (4.2) 渭 mol 路L ~ (-1) of arsenic and inhibited Nrf2 gene expression in cells treated with arsenic for 8 h and 24 h. After treatment with arsenic, the expression of MAPK / ERK was increased by 21.00 渭 mol/L arsenic treatment, and the expression of Bach1N6AMT1MAPK-ERK / ERK mRNA was down-regulated in Nrf2 cells. There was significant difference in the expression of NRF2 gene in HaCaT cells treated with 2.10 渭 mol / L ~ 2.10 渭 mol 路L ~ (-1) P0.001 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) for 24 h. The expression of N6AMT _ (1) -COX-2GSH protein was up-regulated in HaCaT cells treated with arsenic (29.00 渭 mol/L), and the expression of N6AMT1-COX-2GSH protein was down-regulated in HaCaT cells treated with arsenic (29.00 渭 mol / L). Conclusion: 1: 1, arsenic has low promoting effect. 2N6AMT1 can regulate oxidation and oxidation. 3GSH can regulate cell oxidation level. 4 Ros maintain dynamic balance. 5 Bach1 COX-2 can participate in the antioxidant response. 6MAPK / ERK can activate COX-2 expression.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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