Protectin DX对氧化应激状态下人视网膜色素上皮细胞的保护作用及机制的初步研究

发布时间：2018-05-16 04:41

  本文选题：保护素 + 氧化应激　； 参考：《北京协和医学院》2015年博士论文

【摘要】：目的：二十六碳烯酸(Docosahexaenoic acid, DHA)高度富集于视网膜色素上皮(Retinal pigment epithelium, RPE)细胞,经脂氧合酶代谢可被转化为protectin DX(PDX)。本研究目的为确定DHA衍生物PDX是否对氧化应激状态下ARPE-19细胞产生保护作用及其机制,为脂类及其衍生物在年龄相关性黄斑变性防治研究方面提供实验室证据。方法：取生长良好的ARPE-19(第16-18代)细胞,以完全DMEM/F12培养基均匀接种于96孔板(2×104/0.1ml/孔)或六孔板(4×105/2.0ml/孔),每日于倒置显微镜下观察。当细胞生长至接近80%融合时,以无血清DMEM/F12培养基液饥饿细胞24小时。在2000M叔丁基过氧化氢(tert-butylhydroperoxide, t-BH)处理2h对细胞进行氧化应激诱导前,分别以0.05μM、0.5μM、1μM、5μM或10μM PDX预孵育ARPE-19细胞20 mmin。氧化应激诱导结束后,以无血清DMEM/F12培养基液清洗细胞3次,去除多余t-BH。然后以含1%FBS的DMEM/F12培养基液继续处理细胞,按实验不同检测时间点收集细胞或细胞上清样本。其中：1)以含1% FBS的DMEM/F12培养基液处理细胞2小时,收集六孔板中的细胞用于实时荧光定量PCR检测；2)以含1%FBS的DMEM/F12培养基液处理细胞4小时,收集六孔板中细胞用于细胞内ROS检测；3)以含1% FBS的DMEM/F12培养基液处理细胞24小时,收集六孔板中的细胞用于T-AOC和SOD检测及WB分析,或收集96孔板中的细胞上清用于ELISA检测。结果：200μMt-BH诱导ARPE-19细胞2h,细胞内dichlorofluorescein荧光强度较正常对照组显著增强。氧化应激诱导结束后24 h细胞上清VEGF-A蛋白浓度较相同时间点正常对照组VEGF-A浓度增加,差别有统计学意义(P0.001)。5 pMPDX或10μM PDX预孵育ARPE-19细胞20分钟,可显著降低氧化应激诱导后RPE细胞内ROS生成量(P0.01)。以1μM、5μM或10μMPDX预孵育20 mmin,再对ARPE-19细胞进行氧化应激诱导,细胞SOD活性分别是氧化应激模型组的1.28倍、1.37倍和1.48倍,差别有统计学意义(P0.01,P0.01和P0.001)；细胞T-AOC分别是氧化应激模型组的1.40%、1.80%和2.07%,差别有统计学意义(P0.01,P0.001和P0.001)。当PDX预孵育浓度≥0.05μM时,相同时间点培养基上清TNF-α蛋白浓度较氧化应激模型组显著降低,差别有统计学意义(P0.01)。当PDX预孵育浓度≥1μ时,培养基上清VEGF-A蛋白浓度较氧化应激模型组显著降低,差别有统计学意义(P0.01)。当PDX预孵育浓度为10μM时,氧化应激后细胞内PGC-1α蛋白表达较氧化应激模型组细胞PGC-1α蛋白表达显著增加,差别有统计学意义(P0.001)。结论：1) 200μM叔丁基过氧化氢处理ARPE-19细胞2小时,细胞内ROS生成量明显增多,细胞分泌VEGF-A较正常状态下显著增加,细胞活力良好,表现出RPE细胞在经历慢性氧化应激时的典型特征,可作为体外培养的人视网膜色素上皮细胞氧化损伤模型。2) Protectin DX可以显著降低叔丁基过氧化氢诱导的炎症及炎症相关细胞因子释放,并通过直接增加PGC-1α表达而增强细胞的抗氧化能力,进一步调节ROS水平,减轻视网膜色素上皮细胞氧化损伤。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R774.5
，

本文编号：1895520