阻滞Nrf2基因对砷致皮肤角化相关基因及蛋白的影响研究

发布时间：2018-05-08 00:27

  本文选题：混合砷 + HaCaT细胞　； 参考：《新疆医科大学》2017年硕士论文

【摘要】：目的:研究Nrf2基因抑制及无机砷染毒对Nrf2通路相关基因及蛋白表达的影响,探讨其在砷致皮肤细胞氧化应激中的作用,为砷致皮肤损伤提供理论依据。方法:1.慢病毒载体的构建:依据Nrf2基因序列构建Nrf2 sh RNA,重组慢病毒表达载体,并进行测序鉴定。2.慢病毒包装:Keap1抑制质粒大量抽提扩增,共转293T细胞,收集病毒上清,测定病毒滴度。3.用不同的慢病毒载体(Nrf2 shRNA1/2/3和空载体)分别感染HaCaT细胞构建Nrf2基因抑制HaCaT细胞系,感染复数为5,48小时后,用1μg/ml嘌呤霉素筛选稳转株。4.稳转株的鉴定:培养经筛选获得的稳转株,通过实时荧光定量法检测细胞中Nrf2 mRNA表达水平。5.采用MTT还原法检测细胞生长状况,确定LC50及染毒浓度,染毒浓度分别为LC50的1/100、1/50、1/10。6.流式细胞仪检测细胞的凋亡情况。7.RT-PCR检测细胞中Nrf2、Keap1、NF-κB、CBP、As3MT、K-1、K-10、INV、LOR mRNA表达水平。8.ELISA试剂盒检测细胞中HO-1、SAM、HCY、As3MT蛋白表达水平。结果:1.慢病毒干扰载体构建,经基因测序,与目的基因序列完全匹配,慢病毒滴度为2×108TU/ml。2.建立的Nrf2基因抑制稳转细胞株中sh3最好,其基因的沉默效率为87%,作为后续实验细胞。3.混合砷染毒模型细胞48h的LC50为210.00μmol/L,染毒浓度分别为LC50的1/100(2.10μmol/)L,LC50的1/50(4.20μmol/L),LC50的1/10(21.00μmol/L)。4.Keap1基因抑制、2.10μmol/L混合砷染毒促进细胞增殖,细胞生长速度加快;随染毒浓度增加(4.20μmol/L-21.00μmol/L)染毒时间延长(48h、72h)细胞多核细胞出现,脱壁明显。5.与Nrf2抑制对照组相比,8h和24h时2.1μmol/L、4.20μmol/L、21μmol/L浓度组早期凋亡率均升高,且随染毒浓度的增加,凋亡率增加;48h后其早凋亡率均降低,差异均有统计学意义(P0.01)。72h各浓度组早期凋亡率均低于空白对照组,差异有统计学意义(P0.01)。6.与Nrf2抑制对照组及8h相比,Keap1抑制对照组24h、48h、72hKeap1 mRNA表达均下调(P0.01),且随时间延长呈下降趋势;2.1μmol/L、4.2μmol/L、21μmol/L浓度组Keap1 mRNA表达24h、48h均下调(P0.01),表达随浓度增大呈上升趋势。与Nrf2抑制对照组相比,空白对照组和Keap1抑制对照组72hNrf2 mRNA表达均上调(P0.01);2.1μmol/L浓度组24h、48h和72hNrf2 mRNA表达均上调(P0.01);而4.2μmol/L和21μmol/L 24h、72hNrf2 m RNA表达均下调(P0.01)。与空白对照组和Nrf2抑制对照组相比,较低浓度组(2.1、4.20μmol/L)细胞中NF-κB、CBP mRNA的表达上调;高浓度组(21.00μmol/L)长时间(48h、72h)NF-κB、CBP mRNA的表达下调,差异均有统计学意义(P0.01)。各浓度组K-l mRNA在24h、48h时表达较空白对照组和Nrf2抑制对照组下调,72h明显上调,差异有统计学意义(P0.01);与空白对照组和Nrf2抑制对照组相比,较低浓度组(2.10、4.20μmol/L)长时间(48h、72h)K-l0 m RNA的表达上调,高浓度组K-l0 mRNA总体呈下调状态,差异均有统计学意义(P0.01)。与空白对照组和Nrf2抑制对照组相比,较低浓度组(2.10、4.20μmol/L)INV、LOR mRNA 24h和72h表达明显升高,48h表达下调,21μmol/L浓度组INV、LOR m RNA24h后均呈下调状态,差异均有统计学意义(P0.01)。7.与空白对照组和Nrf2抑制对照组相比,4.2μmol/L和21μmol/L混合砷染毒促进As3MT mRNA表达,2.10、4.20μmol/L浓度组8h、24h、48hAs3MT蛋白含量下调,72h表达上调,21μmol/L浓度组长时间48h、72h As3MT蛋白含量上调,差异均有统计学意义(P0.01)。8.与空白对照组相比,Keap1抑制对照组8h、48h和72hSAM蛋白含量均下调(P0.01);Nrf2抑制对照组24h、48h和72hSAM蛋白含量均上调(P0.01);与Nrf2抑制对照组相比,2.1μmol/L、4.2μmol/L、21μmol/L浓度组随时间延长(24h、48h、72h)SAM蛋白含量均下调(P0.01),同时间不同浓度组随浓度升高,呈下降趋势,差异均有统计学意义(P0.01)。与空白对照组和Nrf2抑制对照组相比,8h、24h、48h和72hHCY蛋白含量均上调,差异均有统计学意义(P0.01);空白对照组和Keap1抑制对照组24h后HCY蛋白含量均低于Nrf2抑制对照组,差异有统计学意义(P0.01)。与空白对照组和Nrf2抑制对照组相比,2.10、4.20μmol/L浓度组HO-1蛋白含量由8h、24h的表达上调转至48h、72h表达逐渐下调,21μmol/L浓度组HO-1蛋白含量基本维持下调水平;且均随浓度增加表达逐渐降低,差异均有统计学意义(P0.01)。结论:1.混合砷染毒能引起细胞形态改变,低浓度促进细胞增殖,高浓度促进细胞凋亡。2.Nrf2基因抑制联合混合砷染毒细胞,Nrf2通路相关基因(Nrf2、Keap1、NF-κB、CBP、As3MT、)及蛋白(HO-1、SAM、HCY、As3MT)表达发生改变;角化蛋白基因(K-1、K-10、INV、LOR)表达紊乱,能加速砷诱导的氧化应激反应。3.Nrf2基因抑制下,时间和浓度的交互影响着混合砷对模型细胞的毒性作用,因此,随着染毒时间的延长、染毒浓度的增大,砷暴露对皮肤细胞的氧化损害作用逐渐增大。
[Abstract]:Objective: To study the effect of Nrf2 gene inhibition and arsenic exposure on Nrf2 pathway related genes and protein expression, explore the role of arsenic in the oxidative stress induced by arsenic and provide theoretical basis for arsenic induced skin damage. Method: Construction of 1. lentivirus vector: Construction of Nrf2 sh RNA based on Nrf2 sequence, recombinant lentivirus expression vector, and Sequencing and identification of.2. lentivirus packaging: Keap1 inhibitory plasmids were extracted and amplified, CO converted to 293T cells, collected virus supernatant, and detected virus titer.3. using different lentivirus carriers (Nrf2 shRNA1/2/3 and empty carrier) to construct Nrf2 gene to inhibit HaCaT cell line respectively. The complex number of the infection was 5,48 hours, and 1 mu g/ml purinomycin was used. Identification of stable transfer strain of stable strain.4.: Culture selected stable transgenic plant, detection of Nrf2 mRNA expression level by real-time fluorescence quantitative.5., MTT reduction method was used to detect cell growth status, LC50 and concentration were determined, 1/100,1/50,1/ 10.6. flow cytometry was used to detect cell apoptosis in LC50 1/100,1/50,1/ 10.6. flow cytometer, respectively.7.RT. -PCR detection of Nrf2, Keap1, NF- kappa B, CBP, As3MT, K-1, K-10, INV, LOR mRNA protein expression level. Results: 1. lentivirus interference vector construction, gene sequencing, complete matching with the target gene sequence, the slow virus titer is 2 x In the cell line, SH3 was the best, and its gene silencing efficiency was 87%. As a follow-up experimental cell, the LC50 of 48h was 210 mu mol/L, the concentration of LC50 was LC50 1/100 (2.10 mu mol/) L, LC50 1/50 (4.20 mu mol/L), 2.10 micron (21 micron) gene inhibition, and 2.10 micron mixed arsenic poisoning to promote cell proliferation. The growth rate of cells was accelerated; with increased exposure (4.20 mol/L-21.00 mu mol/L) prolonged (48h, 72h) cell multinuclear cells appeared, the removal of.5. and Nrf2 inhibited the control group, 8h and 24h at 2.1 mol/L, 4.20 mu mol/L, 21 mu mol/L concentration group early apoptosis rate increased, and with the increase of dye concentration, the apoptosis rate increased; after 48H Early apoptosis rate decreased, the difference was statistically significant (P0.01).72h concentration group early apoptosis rate was lower than the blank control group, the difference was statistically significant (P0.01).6. and Nrf2 inhibition control group and 8h, Keap1 inhibited the control group 24h, 48h, 72hKeap1 mRNA expression down (P0.01), and the decrease with time; 2.1 mu, 4.2 Mu The expression of Keap1 mRNA in 21 mol/L concentration group expressed 24h, 48h decreased (P0.01), and the expression increased with the increase of concentration. Compared with the Nrf2 inhibition control group, the expression of 72hNrf2 mRNA expression in the control group and the control group of Keap1 was up up (P0.01), and the 2.1 micron mol/L concentration group was up and up, while 4.2 Mu and 21 Mu were all up. The expression of M RNA was down (P0.01). The expression of NF- kappa B and CBP mRNA in the lower concentration group (2.1,4.20 / mol/L) was up to be up, compared with the blank control group and the Nrf2 inhibition control group. Compared with the blank control group and the Nrf2 control control group, the 72h was obviously up, and the difference was statistically significant (P0.01). Compared with the blank control group and the Nrf2 inhibition control group, the expression of K-l0 m RNA in the lower concentration group (48h, 72h) was up regulated in the lower concentration group (48h, 72h), and the higher concentration group was down down, the difference was statistically significant. Compared with the blank control group and the Nrf2 control control group, the lower concentration group (2.10,4.20 mu mol/L) INV, the LOR mRNA 24h and 72h expression were significantly increased, the 48h expression was down down, the 21 mu mol/L concentration was INV, and the LOR was down down state, the difference was statistically significant, compared with the blank control group and the control control group, 4.2 Mu and 21 micron. The content of 8h, 24h, 48hAs3MT protein was down regulated, the expression of 72h was down regulated, the concentration of 72h was up regulated, the concentration of 72h was up to be up to 48h, and the content of 72h As3MT protein was up regulated in the concentration group of 2.10,4.20 mu mol/L, and the content of 72h As3MT protein was up regulated in 2.10,4.20 mu mol/L concentration group. RF2 inhibition group 24h, 48h and 72hSAM protein content up up (P0.01), compared with the Nrf2 control control group, 2.1 mu mol/L, 4.2 mu mol/L, 21 micron concentration group decreased with time (24h, 48h, 72h) decreased (24h, 48h, 72h) decreased with the concentration, the difference was statistically significant. The content of 8h, 24h, 48h and 72hHCY increased in both group and Nrf2 control group, and the difference was statistically significant (P0.01). The content of HCY protein in the blank control group and the Keap1 inhibition control group was lower than that of the Nrf2 inhibition control group, and the difference was statistically significant (P0.01). The content of HO-1 protein in the group was up to 48h, the expression of 24h was down to 48h, the expression of 72h decreased gradually. The content of HO-1 protein in the concentration group of 21 u mol/L was basically down regulated, and the expression gradually decreased with the increase of concentration. The difference was statistically significant (P0.01). Conclusion: 1. mixed arsenic poisoning can lead to cell morphology change, low concentration promote cell proliferation and high concentration. The expression of Nrf2 pathway related genes (Nrf2, Keap1, NF- kappa B, CBP, As3MT,) and protein (HO-1, SAM, HCY, As3MT) expression changes, the expression disorder of the protein gene (HO-1, SAM, HCY, As3MT), and the inhibition of the time and concentration of arsenic induced oxidative stress by the gene inhibition of arsenic induced.2.Nrf2 genes. The toxic effects of mixed arsenic on the model cells were influenced by each other. Therefore, the oxidative damage of skin cells increased gradually with the prolongation of the exposure time and the increase of the concentration of arsenic.

【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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