【摘要】：背景:紫外線輻射是導(dǎo)致急、慢性皮膚損傷,皮膚光老化甚至皮膚腫瘤的的一個主要的環(huán)境因素。它主要是通過激活相關(guān)信號通路誘導(dǎo)基因的表達(dá)和一些蛋白的重新編程,從而參與皮膚疾病發(fā)生發(fā)展的過程。真核翻譯起始因子eIF2α是參與這一過程的主要的調(diào)控因子,eIF2α磷酸化能夠抑制細(xì)胞內(nèi)大量蛋白的合成,選擇性的激活某些蛋白的合成,從而減輕內(nèi)質(zhì)網(wǎng)負(fù)荷。但是過量的內(nèi)質(zhì)網(wǎng)應(yīng)激會誘導(dǎo)eIF2α磷酸化激活Chop和Caspase家族蛋白的表達(dá)最終誘導(dǎo)細(xì)胞凋亡。eIF2α磷酸化是生物適應(yīng)環(huán)境變化的一種有效的方式,在應(yīng)對外界刺激過程中起到了重要的作用。UVA能夠誘導(dǎo)質(zhì)網(wǎng)應(yīng)激,激活PERK磷酸化從而誘導(dǎo)其下游eIF2α發(fā)生磷酸化起到保護(hù)的作用。另外UVA輻射做為一種氧化應(yīng)激能夠誘導(dǎo)ROS的產(chǎn)生,引起氧化損傷激活一系列的抗氧化通路的激活,其中重要的通路之一是誘導(dǎo)Nrf2-HO-1抗氧化通路。UVA能夠激活Nrf2并且促進(jìn)其轉(zhuǎn)核,結(jié)合抗氧化反應(yīng)原件(ARE)促進(jìn)下游的抗氧化酶(HO-1、SOD、NQO1等)Ⅱ期解毒酶的合成。其中血紅素氧合酶HO-1是催化血紅素的起始酶和限速酶,它能夠催化血紅素,產(chǎn)生具有抗氧、抗炎作用的鐵蛋白,一氧化碳(CO)以及膽紅素。已有的研究表明UVA能夠誘導(dǎo)eIF2α磷酸化以及激活Nrf2-HO-1通路的表達(dá)兩種保護(hù)機制,然而目前關(guān)于對于內(nèi)質(zhì)網(wǎng)應(yīng)激和氧化應(yīng)激之間的關(guān)系研究較少,且對于兩條信號通路之間的作用機制也不十分清楚。因此研究eIF2α磷酸化在UVA誘導(dǎo)的氧化損傷中Nrf2-HO-1表達(dá)中的作用以及其對UVA誘導(dǎo)的氧化損傷中的保護(hù)作用具有重要意義。目的:采用不同劑量的UVA(50、100、150、200、250 kJ/m2)輻射小鼠角質(zhì)形成細(xì)胞(JB6),檢測UVA的氧化損傷作用;驗證UVA能夠誘導(dǎo)eIF2α磷酸化,驗證UVA能夠激活Nrf2-HO-1通路;驗證的Salubrinal,GSK2606414對eIF2α磷酸化的作用并篩選出最佳的藥物濃度;探索3μM的Salubrinal,0.5μM的GSK2606414分別預(yù)處理對150kJ/m2UVA輻射細(xì)胞對Nrf2,Bach1,H0-1 mRNA的影響;探索Salubrinal,GSK2606414分別預(yù)處理對150 kJ/m2UVA輻射細(xì)胞后Grp78,Nrf2,Bach1,HO-1,P-eIF2α,eIF2α蛋白水平的影響;探索eIF2α磷酸化的改變對細(xì)胞形態(tài),細(xì)胞活力,細(xì)胞內(nèi)ROS的水平以及細(xì)胞周期的影響,探討在UVA造成的氧化損傷中eIF2α磷酸化的保護(hù)作用。研究方法:采用:mts檢測uva對jb6細(xì)胞活力的影響;fitc-dapi免疫熒光染色法檢測不同劑量的uva對jb6細(xì)胞形態(tài)的影響;dcfh-da熒光探針檢測eif2α磷酸化改變對細(xì)胞內(nèi)ros的影響;實時定量pcr檢測nrf2、bach1、ho-1的表達(dá)的影響;westernblotting檢測grp78、nrf2、bach1、ho-1、p-eif2α、eif2α蛋白表達(dá)的改變;流式細(xì)胞術(shù)檢測eif2α磷酸化改變對細(xì)胞周期的影響。結(jié)果:1.長波紫外線uva輻射能夠誘導(dǎo)jb6細(xì)胞氧化損傷,且損傷程度呈劑量依賴性:與未輻射組相比,細(xì)胞形態(tài),細(xì)胞活力在小劑量50kj/m2uva輻射時無明顯的影響(p0.05)。100、150、200、250kj/m2uva輻射之后隨劑量的增加細(xì)胞碎片增多,細(xì)胞活力顯著性降低且具有統(tǒng)計學(xué)差異(p0.05)。2.uva輻射誘導(dǎo)eif2α磷酸化呈劑量和時間依賴性:與未輻射組相比,不同劑量的uva(50、100、150、200、250kj/m2)能夠誘導(dǎo)eif2α磷酸化,磷酸化程度隨著uva劑量的增加而增高,在250kj/m2的劑量輻射較200kj/m2的磷酸化程度稍微有所降低。因此我們選擇中等生理劑量的150kj/m2uva輻射jb6細(xì)胞檢測不同時間點eif2α的磷酸化情況,與未輻射組相比,uva輻射之后eif2α磷酸化即刻增加且程度最高,隨著時間的延長磷酸化程度下降。3.uva輻射能夠激活nrf2-are通路且呈時間和劑量依賴性:與未輻射組相比,隨著uva劑量的增加,nrf2蛋白水平顯著增加,但250kj/m2uva輻射有所降低。而ho-1蛋白水平隨著uva劑量的增加而顯著增加。選擇150kj/m2uva輻射jb6細(xì)胞后檢測不同時間點nrf2,ho-1基因和蛋白水平的改變,nrf2,ho-1的轉(zhuǎn)錄水平隨著時間點的增加而增加nrf2在4h達(dá)到高峰,ho-1在12h達(dá)到最高峰。nrf2,ho-1的蛋白水平先增加后降低,ho-1的蛋白水平在12h達(dá)到高峰。4.salubrinal,gsk2606414分別預(yù)處理對150kj/m2uva輻射jb6細(xì)胞eif2α磷酸化的影響:與對照組相比,salubrinal預(yù)處理對細(xì)胞活力未發(fā)現(xiàn)顯著性影響(p0.05)。與對照組相比,不同濃度的gsk2606414預(yù)處理細(xì)胞活力未發(fā)現(xiàn)顯著性影響(p0.05)。不同濃度salubrinal預(yù)處理對150kj/m2uva輻射jb6細(xì)胞eif2α的磷酸化具有增強的作用呈現(xiàn)出劑量的依賴性,但在5μm的濃度下較3μm組eif2α的磷酸化稍微有所降低。不同濃度gsk2606414預(yù)處理對150kj/m2uva輻射jb6細(xì)胞eif2α的磷酸化具有抑制作用,在0.5μm時磷酸化程度最低。5.eif2α磷酸化的改變對nrf2、bach1、ho-1等rna和蛋白的影響:3μM Salubrinal預(yù)處理聯(lián)合150 k J/m2UVA輻射與單獨150 kJ/m2UVA組相比eIF2α磷酸化增加,0.5μM GSK2606414預(yù)處理聯(lián)合150 kJ/m2UVA輻射與單獨150kJ/m2UVA組相比eIF2α磷酸化降低。Salubrinal聯(lián)合UVA組較單獨UVA輻射組對Bach1,Nrf2,HO-1基因水平影響不大,可以促進(jìn)Grp78蛋白表達(dá),、誘導(dǎo)Nrf2、HO-1蛋白表達(dá)延遲。GSK2606414聯(lián)合UVA組較單獨UVA組延遲Grp78表達(dá),誘導(dǎo)Nrf2,HO-1蛋白表達(dá)提前。6.eIF2α磷酸化的改變對細(xì)胞生物學(xué)的影響:與未輻射組相比,3μM Salubrinal預(yù)處理,0.5μM GSK2606414預(yù)處理對細(xì)胞活力無顯著性影響。與150 kJ/m2UVA輻射組相比,3μM Salubrinal預(yù)處理聯(lián)合150kJ/m2UVA輻射細(xì)胞的活力增加,0.5μM GSK2606414預(yù)處理聯(lián)合150 kJ/m2UVA輻射細(xì)胞的活力降低。與未輻射組相比,3μM Salubrinal預(yù)處理,0.5μM GSK2606414預(yù)處理對細(xì)胞周期無顯著性影響,與150 kJ/m2UVA輻射組相比,3μM Salubrinal預(yù)處理聯(lián)合150 kJ/m2UVA輻射細(xì)胞周期S期增加7.28±1.03(P0.05),0.5μM GSK2606414預(yù)處理聯(lián)合150 kJ/m2UVA輻射細(xì)胞的周期S期降低差異不顯著。結(jié)論:UVA輻射能夠激活內(nèi)質(zhì)網(wǎng)應(yīng)激,誘導(dǎo)eIF2α磷酸化且eIF2α磷酸化呈現(xiàn)出劑量和時間的依賴性。UVA輻射作為一種氧化應(yīng)激也能夠誘導(dǎo)Nrf2-ARE通路的激活且也呈現(xiàn)出劑量和時間的依賴性。使用Sal預(yù)處理能夠維持eIF2α磷酸化,使用GSK2606414能夠抑制eIF2α磷酸化。eIF2α磷酸化參與Nrf2-HO-1通路的調(diào)控。維持eIF2α磷酸化的增加能夠減輕UVA誘導(dǎo)的氧化損傷,促進(jìn)細(xì)胞的增殖。而抑制eIF2α磷酸化會加重UVA誘導(dǎo)的氧化損傷抑制細(xì)胞的增殖。
[Abstract]:BACKGROUND: UV radiation is a major environmental factor leading to acute, chronic skin lesions, skin photoaging, and even skin tumors. It is mainly the process of participating in the development of the skin disease by activating the expression of the related signal path and the reprogramming of some proteins. EIF2 is the main control factor involved in this process, and the phosphorylation of eIF2 can inhibit the synthesis of a large amount of protein in the cell, selectively activate the synthesis of certain proteins, thereby reducing the load of the endoplasmic reticulum. However, excessive endoplasmic reticulum stress induces the expression of eIF2 and the expression of Caspase family proteins to induce apoptosis. The phosphorylation of eIF2 is an effective way to adapt to the change of the environment, and plays an important role in the process of external stimulation. UVA can induce stress, activate the phosphorylation of PERK, and induce the phosphorylation of eIF2 in its downstream. In addition, UVA radiation is an oxidative stress that can induce ROS production, which causes oxidative damage to activate a series of activation of the antioxidant pathway, one of which is the induction of the Nrf2-HO-1 antioxidant pathway. UVA can activate Nrf2 and promote its transformation, and combine the anti-oxidation reaction original (ARE) to promote the synthesis of the downstream anti-oxidation enzyme (HO-1, SOD, NQO1, etc.) phase II detoxification enzyme. Wherein the heme oxygenase HO-1 is a starting enzyme and a speed-limiting enzyme for catalyzing the heme, which can catalyze the heme to generate ferritin, carbon monoxide (CO) and bilirubin with anti-oxygen and anti-inflammatory effects. Studies have shown that UVA can induce the phosphorylation of eIF2 and the activation of two mechanisms of protection of the Nrf2-HO-1 pathway, however, there are few studies on the relationship between endoplasmic reticulum stress and oxidative stress, and the mechanism of action between the two signal pathways is not clear. Therefore, it is of great significance to study the role of eIF2 in the expression of Nrf2-HO-1 in the oxidative damage induced by UVA and its protective effect on the oxidative damage induced by UVA. Objective: To study the effects of UVA (50,100,150,200,250 kJ/ m2) on the oxidative damage of UVA, and to verify that UVA can induce the phosphorylation of eIF2 and to verify that UVA can activate Nrf2-HO-1 pathway. The effects of pre-treatment on Nrf2, Bach1, H0-1 mRNA of 150 kJ/ m2UVA radiation cells and the effect of the pretreatment on the level of Grp78, Nrf2, Bach1, HO-1, P-eIF2 and eIF2 in the radiation cells of 150 kJ/ m2UVA were respectively pre-treated with 3. m u.M of Salubrinal and 0.5. m u.M of GSK2606414. The changes of the phosphorylation of eIF2 to the cell morphology were explored. The cell activity, the level of ROS in the cell and the effect of cell cycle were discussed, and the protective effect of eIF2 on the phosphorylation of eIF2 in the oxidative damage caused by UVA was discussed. Methods: the effect of uva on the activity of jb6 cells was detected by the method of mts; the effect of uva on the morphology of jb6 cells was detected by the fitc-dapi immunofluorescent staining method; the effect of the changes of the ethec-da fluorescent probe on the ros in the cells was detected by the dcfh-da fluorescent probe; the effect of nrf2, bachi1, ho-1 was detected in real time by the real-time quantitative pcr. The changes of the expression of grp78, nrf2, bachi1, ho-1, p-eif2, and eif2 were detected by the western blotting, and the effect of the alteration of eisf2 on the cell cycle was detected by flow cytometry. Results:1. long-wave ultraviolet uva radiation can induce the oxidative damage of jb6 cells, and the degree of damage is dose-dependent: compared with the non-radiation group, the cell morphology and the cell viability do not have a significant effect on the dose of 50 kj/ m2uva radiation (p0.05).100,150,200,250 kj/ m2uva radiation, The significant decrease in cell viability and statistically significant (p0.05) .2.uva radiation induced the dose and time-dependent phosphorylation of eif2: the different doses of uva (50,100,150,200,250 kj/ m2) were able to induce esif2 phosphorylation, and the degree of phosphorylation increased with the increase of the uva dose, The level of phosphorylation at 250 kj/ m2 was slightly lower than the level of phosphorylation of 200 kj/ m2. Therefore, we selected a moderate physiological dose of 150 kj/ m2uva radiation jb6 cells to detect the phosphorylation of eif2 antigen at different time points, and the phosphorylation of eif2 was increased immediately after uva irradiation compared to the non-irradiated group, The decrease of .3.uva radiation over time allows for the activation of the nrf2-are pathway and in time and dose-dependent: as the uva dose increases, the nrf2 protein level increases significantly as the uva dose increases, but the 250 kj/ m2uva radiation is reduced. While the level of ho-1 protein increased significantly with the increase of the uva dose. The changes of nrf2, ho-1 and protein levels of nrf2, ho-1 at different time points were detected after the selection of 150 kj/ m2uva radiation jb6 cells, and the transcription level of nrf2, ho-1 reached the peak at 4 h with the increase of the time point, and ho-1 reached its peak at 12 h. The protein level of nrf2 and ho-1 decreased firstly, and the protein level of ho-1 reached a peak at 12 h. Compared with the control group, no significant effect was found on the activity of the pre-treated cells of gsk2606414 in different concentrations (p0.05). Phosphorylation of the eif2 cells of the 150kj/ m2uva radiation jb6 cells with different concentrations of salubrinal pretreatment showed a dose-dependent manner, but the phosphorylation of eif2 cells at a concentration of 5. m u.m was slightly reduced. the pre-treatment of gsk2606414 at different concentrations had an inhibitory effect on the phosphorylation of eif2 cells of the 150kj/ m2uva radiation jb6 cells, the lowest phosphorylation of 5. m The effect of ho-1, such as rna and protein, was that the phosphorylation of eIF2 was increased with the addition of 150 kJ/ m2UVA radiation compared with the 150 kJ/ m2UVA group alone, and the phosphorylation of eIF2 was reduced by 0.5. m The effect of UVA radiation on Bach1, Nrf2 and HO-1 gene level in the combined UVA group was not significant, and the expression of Grp78 protein could be promoted, and the expression of Nrf2 and HO-1 protein could be induced. GSK2606414 delayed the expression of Grp78 in the UVA group and induced Nrf2 and HO-1 protein expression in advance. Compared with the 150 kJ/ m2UVA radiation group, the activity of the 3. m M Salubrinal pretreatment combined with 150 kJ/ m2UVA radiation cells increased, and the activity of the 0.5. mu.M GSK2606414 pre-treatment combined with 150 kJ/ m2UVA radiation cells was reduced. in contrast to that non-radiation group, the pre-treatment of 3. mu. M Salubrinal pretreatment, 0.5. mu.M GSK2606414 pre-treatment had no significant effect on the cell cycle, Compared with the 150 kJ/ m2UVA radiation group, the cell cycle S phase of the combined 150kJ/ m2UVA radiation cell increased by 7.28-1.03 (P0.05), and the difference between the pre-treatment of 0.5 & mu; M GSK2606414 and the cycle S phase of the 150 kJ/ m2UVA radiation cell was not significant. Conclusion: UVA radiation can activate endoplasmic reticulum stress, induce eIF2-induced phosphorylation, and eIF2-induced phosphorylation presents a dose-and time-dependent dependence. UVA radiation as an oxidative stress can also induce activation of the Nrf2-ARE pathway and also exhibit a dose and time-dependent dependence. The phosphorylation of eIF2 was maintained by using the Sal pretreatment, and the phosphorylation of eIF2 was inhibited using GSK2606414. The phosphorylation of eIF2 is involved in the regulation of Nrf2-HO-1 pathway. The increased phosphorylation of eIF2 can reduce the oxidative damage induced by UVA and promote cell proliferation. While inhibiting the phosphorylation of eif2 will increase the proliferation of uva-induced oxidative damage-inhibiting cells.
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R751

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1 薛方方;eIF2α磷酸化對長波紫外線誘導(dǎo)的小鼠角質(zhì)形成細(xì)胞氧化損傷中Nrf2-HO-1通路的影響[D];重慶大學(xué);2016年

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