本文選題：混合砷 + HaCaT細胞�。� 參考：《新疆醫(yī)科大學》2017年碩士論文

【摘要】：目的:研究Nrf2基因抑制及無機砷染毒對Nrf2通路相關基因及蛋白表達的影響,探討其在砷致皮膚細胞氧化應激中的作用,為砷致皮膚損傷提供理論依據(jù)。方法:1.慢病毒載體的構(gòu)建:依據(jù)Nrf2基因序列構(gòu)建Nrf2 sh RNA,重組慢病毒表達載體,并進行測序鑒定。2.慢病毒包裝:Keap1抑制質(zhì)粒大量抽提擴增,共轉(zhuǎn)293T細胞,收集病毒上清,測定病毒滴度。3.用不同的慢病毒載體(Nrf2 shRNA1/2/3和空載體)分別感染HaCaT細胞構(gòu)建Nrf2基因抑制HaCaT細胞系,感染復數(shù)為5,48小時后,用1μg/ml嘌呤霉素篩選穩(wěn)轉(zhuǎn)株。4.穩(wěn)轉(zhuǎn)株的鑒定:培養(yǎng)經(jīng)篩選獲得的穩(wěn)轉(zhuǎn)株,通過實時熒光定量法檢測細胞中Nrf2 mRNA表達水平。5.采用MTT還原法檢測細胞生長狀況,確定LC50及染毒濃度,染毒濃度分別為LC50的1/100、1/50、1/10。6.流式細胞儀檢測細胞的凋亡情況。7.RT-PCR檢測細胞中Nrf2、Keap1、NF-κB、CBP、As3MT、K-1、K-10、INV、LOR mRNA表達水平。8.ELISA試劑盒檢測細胞中HO-1、SAM、HCY、As3MT蛋白表達水平。結(jié)果:1.慢病毒干擾載體構(gòu)建,經(jīng)基因測序,與目的基因序列完全匹配,慢病毒滴度為2×108TU/ml。2.建立的Nrf2基因抑制穩(wěn)轉(zhuǎn)細胞株中sh3最好,其基因的沉默效率為87%,作為后續(xù)實驗細胞。3.混合砷染毒模型細胞48h的LC50為210.00μmol/L,染毒濃度分別為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混合砷染毒促進細胞增殖,細胞生長速度加快;隨染毒濃度增加(4.20μmol/L-21.00μmol/L)染毒時間延長(48h、72h)細胞多核細胞出現(xiàn),脫壁明顯。5.與Nrf2抑制對照組相比,8h和24h時2.1μmol/L、4.20μmol/L、21μmol/L濃度組早期凋亡率均升高,且隨染毒濃度的增加,凋亡率增加;48h后其早凋亡率均降低,差異均有統(tǒng)計學意義(P0.01)。72h各濃度組早期凋亡率均低于空白對照組,差異有統(tǒng)計學意義(P0.01)。6.與Nrf2抑制對照組及8h相比,Keap1抑制對照組24h、48h、72hKeap1 mRNA表達均下調(diào)(P0.01),且隨時間延長呈下降趨勢;2.1μmol/L、4.2μmol/L、21μmol/L濃度組Keap1 mRNA表達24h、48h均下調(diào)(P0.01),表達隨濃度增大呈上升趨勢。與Nrf2抑制對照組相比,空白對照組和Keap1抑制對照組72hNrf2 mRNA表達均上調(diào)(P0.01);2.1μmol/L濃度組24h、48h和72hNrf2 mRNA表達均上調(diào)(P0.01);而4.2μmol/L和21μmol/L 24h、72hNrf2 m RNA表達均下調(diào)(P0.01)。與空白對照組和Nrf2抑制對照組相比,較低濃度組(2.1、4.20μmol/L)細胞中NF-κB、CBP mRNA的表達上調(diào);高濃度組(21.00μmol/L)長時間(48h、72h)NF-κB、CBP mRNA的表達下調(diào),差異均有統(tǒng)計學意義(P0.01)。各濃度組K-l mRNA在24h、48h時表達較空白對照組和Nrf2抑制對照組下調(diào),72h明顯上調(diào),差異有統(tǒng)計學意義(P0.01);與空白對照組和Nrf2抑制對照組相比,較低濃度組(2.10、4.20μmol/L)長時間(48h、72h)K-l0 m RNA的表達上調(diào),高濃度組K-l0 mRNA總體呈下調(diào)狀態(tài),差異均有統(tǒng)計學意義(P0.01)。與空白對照組和Nrf2抑制對照組相比,較低濃度組(2.10、4.20μmol/L)INV、LOR mRNA 24h和72h表達明顯升高,48h表達下調(diào),21μmol/L濃度組INV、LOR m RNA24h后均呈下調(diào)狀態(tài),差異均有統(tǒng)計學意義(P0.01)。7.與空白對照組和Nrf2抑制對照組相比,4.2μmol/L和21μmol/L混合砷染毒促進As3MT mRNA表達,2.10、4.20μmol/L濃度組8h、24h、48hAs3MT蛋白含量下調(diào),72h表達上調(diào),21μmol/L濃度組長時間48h、72h As3MT蛋白含量上調(diào),差異均有統(tǒng)計學意義(P0.01)。8.與空白對照組相比,Keap1抑制對照組8h、48h和72hSAM蛋白含量均下調(diào)(P0.01);Nrf2抑制對照組24h、48h和72hSAM蛋白含量均上調(diào)(P0.01);與Nrf2抑制對照組相比,2.1μmol/L、4.2μmol/L、21μmol/L濃度組隨時間延長(24h、48h、72h)SAM蛋白含量均下調(diào)(P0.01),同時間不同濃度組隨濃度升高,呈下降趨勢,差異均有統(tǒng)計學意義(P0.01)。與空白對照組和Nrf2抑制對照組相比,8h、24h、48h和72hHCY蛋白含量均上調(diào),差異均有統(tǒng)計學意義(P0.01);空白對照組和Keap1抑制對照組24h后HCY蛋白含量均低于Nrf2抑制對照組,差異有統(tǒng)計學意義(P0.01)。與空白對照組和Nrf2抑制對照組相比,2.10、4.20μmol/L濃度組HO-1蛋白含量由8h、24h的表達上調(diào)轉(zhuǎn)至48h、72h表達逐漸下調(diào),21μmol/L濃度組HO-1蛋白含量基本維持下調(diào)水平;且均隨濃度增加表達逐漸降低,差異均有統(tǒng)計學意義(P0.01)。結(jié)論:1.混合砷染毒能引起細胞形態(tài)改變,低濃度促進細胞增殖,高濃度促進細胞凋亡。2.Nrf2基因抑制聯(lián)合混合砷染毒細胞,Nrf2通路相關基因(Nrf2、Keap1、NF-κB、CBP、As3MT、)及蛋白(HO-1、SAM、HCY、As3MT)表達發(fā)生改變;角化蛋白基因(K-1、K-10、INV、LOR)表達紊亂,能加速砷誘導的氧化應激反應。3.Nrf2基因抑制下,時間和濃度的交互影響著混合砷對模型細胞的毒性作用,因此,隨著染毒時間的延長、染毒濃度的增大,砷暴露對皮膚細胞的氧化損害作用逐漸增大。
[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.

【學位授予單位】：新疆醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R114

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