本文選題：保護(hù)素 + 氧化應(yīng)激��； 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：目的：二十六碳烯酸(Docosahexaenoic acid, DHA)高度富集于視網(wǎng)膜色素上皮(Retinal pigment epithelium, RPE)細(xì)胞,經(jīng)脂氧合酶代謝可被轉(zhuǎn)化為protectin DX(PDX)。本研究目的為確定DHA衍生物PDX是否對(duì)氧化應(yīng)激狀態(tài)下ARPE-19細(xì)胞產(chǎn)生保護(hù)作用及其機(jī)制,為脂類及其衍生物在年齡相關(guān)性黃斑變性防治研究方面提供實(shí)驗(yàn)室證據(jù)。方法：取生長良好的ARPE-19(第16-18代)細(xì)胞,以完全DMEM/F12培養(yǎng)基均勻接種于96孔板(2×104/0.1ml/孔)或六孔板(4×105/2.0ml/孔),每日于倒置顯微鏡下觀察。當(dāng)細(xì)胞生長至接近80%融合時(shí),以無血清DMEM/F12培養(yǎng)基液饑餓細(xì)胞24小時(shí)。在2000M叔丁基過氧化氫(tert-butylhydroperoxide, t-BH)處理2h對(duì)細(xì)胞進(jìn)行氧化應(yīng)激誘導(dǎo)前,分別以0.05μM、0.5μM、1μM、5μM或10μM PDX預(yù)孵育ARPE-19細(xì)胞20 mmin。氧化應(yīng)激誘導(dǎo)結(jié)束后,以無血清DMEM/F12培養(yǎng)基液清洗細(xì)胞3次,去除多余t-BH。然后以含1%FBS的DMEM/F12培養(yǎng)基液繼續(xù)處理細(xì)胞,按實(shí)驗(yàn)不同檢測(cè)時(shí)間點(diǎn)收集細(xì)胞或細(xì)胞上清樣本。其中：1)以含1% FBS的DMEM/F12培養(yǎng)基液處理細(xì)胞2小時(shí),收集六孔板中的細(xì)胞用于實(shí)時(shí)熒光定量PCR檢測(cè)；2)以含1%FBS的DMEM/F12培養(yǎng)基液處理細(xì)胞4小時(shí),收集六孔板中細(xì)胞用于細(xì)胞內(nèi)ROS檢測(cè)；3)以含1% FBS的DMEM/F12培養(yǎng)基液處理細(xì)胞24小時(shí),收集六孔板中的細(xì)胞用于T-AOC和SOD檢測(cè)及WB分析,或收集96孔板中的細(xì)胞上清用于ELISA檢測(cè)。結(jié)果：200μMt-BH誘導(dǎo)ARPE-19細(xì)胞2h,細(xì)胞內(nèi)dichlorofluorescein熒光強(qiáng)度較正常對(duì)照組顯著增強(qiáng)。氧化應(yīng)激誘導(dǎo)結(jié)束后24 h細(xì)胞上清VEGF-A蛋白濃度較相同時(shí)間點(diǎn)正常對(duì)照組VEGF-A濃度增加,差別有統(tǒng)計(jì)學(xué)意義(P0.001)。5 pMPDX或10μM PDX預(yù)孵育ARPE-19細(xì)胞20分鐘,可顯著降低氧化應(yīng)激誘導(dǎo)后RPE細(xì)胞內(nèi)ROS生成量(P0.01)。以1μM、5μM或10μMPDX預(yù)孵育20 mmin,再對(duì)ARPE-19細(xì)胞進(jìn)行氧化應(yīng)激誘導(dǎo),細(xì)胞SOD活性分別是氧化應(yīng)激模型組的1.28倍、1.37倍和1.48倍,差別有統(tǒng)計(jì)學(xué)意義(P0.01,P0.01和P0.001)；細(xì)胞T-AOC分別是氧化應(yīng)激模型組的1.40%、1.80%和2.07%,差別有統(tǒng)計(jì)學(xué)意義(P0.01,P0.001和P0.001)。當(dāng)PDX預(yù)孵育濃度≥0.05μM時(shí),相同時(shí)間點(diǎn)培養(yǎng)基上清TNF-α蛋白濃度較氧化應(yīng)激模型組顯著降低,差別有統(tǒng)計(jì)學(xué)意義(P0.01)。當(dāng)PDX預(yù)孵育濃度≥1μ時(shí),培養(yǎng)基上清VEGF-A蛋白濃度較氧化應(yīng)激模型組顯著降低,差別有統(tǒng)計(jì)學(xué)意義(P0.01)。當(dāng)PDX預(yù)孵育濃度為10μM時(shí),氧化應(yīng)激后細(xì)胞內(nèi)PGC-1α蛋白表達(dá)較氧化應(yīng)激模型組細(xì)胞PGC-1α蛋白表達(dá)顯著增加,差別有統(tǒng)計(jì)學(xué)意義(P0.001)。結(jié)論：1) 200μM叔丁基過氧化氫處理ARPE-19細(xì)胞2小時(shí),細(xì)胞內(nèi)ROS生成量明顯增多,細(xì)胞分泌VEGF-A較正常狀態(tài)下顯著增加,細(xì)胞活力良好,表現(xiàn)出RPE細(xì)胞在經(jīng)歷慢性氧化應(yīng)激時(shí)的典型特征,可作為體外培養(yǎng)的人視網(wǎng)膜色素上皮細(xì)胞氧化損傷模型。2) Protectin DX可以顯著降低叔丁基過氧化氫誘導(dǎo)的炎癥及炎癥相關(guān)細(xì)胞因子釋放,并通過直接增加PGC-1α表達(dá)而增強(qiáng)細(xì)胞的抗氧化能力,進(jìn)一步調(diào)節(jié)ROS水平,減輕視網(wǎng)膜色素上皮細(xì)胞氧化損傷。
[Abstract]:Objective: Docosahexaenoic acid (Docosahexaenoic acid, DHA) is highly enriched in retinal pigment epithelium (Retinal pigment epithelium, RPE) cells. The metabolism of lipoxygenase can be converted to protectin DX (PDX). Laboratory evidence was provided for the study of lipid and its derivatives in the prevention and control of age-related macular degeneration. Methods: a well grown ARPE-19 (16-18 generation) cell was inoculated evenly on the 96 orifice (2 x 104/0.1ml/ hole) or six orifice (4 x 105/2.0ml/ hole) on a complete DMEM/F12 medium. Near 80% fusion, the serum-free DMEM/F12 medium was used for 24 hours. Before the oxidative stress induced by 2000M 2H Ding Ji hydrogen peroxide (tert-butylhydroperoxide, t-BH), the cells were induced by 0.05 mu M, 0.5 mu M, 1 mu M, 5 u M or 10 micron M PDX incubating ARPE-19 cells 20 for oxidative stress induction. 2 culture medium cleaning cells 3 times, remove redundant t-BH. and then continue to treat cells with 1%FBS DMEM/F12 culture medium, collect cells or cell supernatant samples according to different test time points. 1) with 1% FBS DMEM/F12 medium solution for 2 hours, and collect six Kong Banzhong cells for real-time fluorescence quantitative PCR detection; 2) the cells were treated with 1%FBS DMEM/F12 medium solution for 4 hours, and the cells in the six pore plate were used for intracellular ROS detection; 3) the cells were treated with 1% FBS DMEM/F12 medium for 24 hours, and the cells in the six pore plate were used for T-AOC and SOD detection and WB analysis, or the cell supernatant in 96 orifice plates was used for ELISA detection. The result: 200 micron Mt-BH The fluorescence intensity of dichlorofluorescein in ARPE-19 cells was significantly enhanced. The concentration of VEGF-A protein in the 24 h cell supernatant was higher than that of the normal control group after the induction of oxidative stress. The concentration of VEGF-A increased in the normal control group. The difference was statistically significant (P0.001).5 pMPDX or 10 micron M PDX incubation for 20 minutes, which was significant for 20 minutes. The amount of ROS formation (P0.01) in RPE cells was reduced after oxidative stress. 20 mmin was incubated with 1 u M, 5 mu M or 10 u MPDX, and ARPE-19 cells were induced by oxidative stress. The SOD activity of the cells was 1.28 times, 1.37 times and 1.48 times of that of the oxidative stress model group, and the difference was statistically significant (P0.01, P0.01 and P0.001). The difference was statistically significant (P0.01, P0.001 and P0.001) in the model group (P0.01, P0.001 and P0.001). When PDX preincubation concentration was more than 0.05 mu M, the concentration of TNF- alpha protein in the same time point culture medium was significantly lower than that of the oxidative stress model group. The difference was statistically significant (P0.01). When the pre incubation concentration of PDX was more than 1 mu, the concentration of VEGF-A protein in the culture medium was compared. The oxidative stress model group decreased significantly (P0.01). When the pre incubation concentration of PDX was 10 u M, the expression of PGC-1 alpha protein in cells after oxidative stress increased significantly compared with the oxidative stress model group PGC-1 alpha protein expression, the difference was statistically significant (P0.001). Conclusion: 1) 200 u M tert butyl peroxide treated ARPE-19 cells 2 small The production of ROS in cells increased significantly, and the cell secretion of VEGF-A increased significantly under the normal state, and the cell viability was good. The typical characteristics of RPE cells during the chronic oxidative stress could be used as the oxidative damage model of the cultured human retinal pigment epithelial cells in vitro. Protectin DX could significantly reduce the tert butyl peroxide. Induced inflammatory and inflammatory cytokines release, and enhance the antioxidant capacity of cells by directly increasing the expression of PGC-1 alpha, further regulating the level of ROS and alleviating the oxidative damage of retinal pigment epithelial cells.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R774.5
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本文編號(hào)：1895520