过氧化氢通过MAPKs通路促进巨噬细胞中Angptl4的表达

发布时间：2018-06-05 11:15

  本文选题：血管生成素样蛋白4(Angptl4) + 过氧化氢(H_2O_2)　； 参考：《山东大学》2017年硕士论文

【摘要】：血管生成素样蛋白4(Angiopoietin-like protein 4,Angptpl4)自被发现以来,其在疾病中的作用得到越来越多的研究和重视。现已有研究表明Angpt14参与动脉粥样硬化的发生发展。但是其确切的作用机制仍不清楚。同样地,大量的研究表明氧化应激与心血管疾病密切相关,但其发挥作用的途径还需要进一步探究。随着研究的深入,氧化应激与炎症反应在动脉粥样硬疾病中所起的作用进一步被揭示,已经成为探究其发病机制及寻找新的治疗手段的研究重点。研究目的:本实验旨在探究过氧化氢(hydrogen peroxide,H_2O_2)对小鼠巨噬细胞株RAW264.7中血管生成素样蛋白4(Angptl4)水平的影响以及进一步探究参与H_2O_2调节Angpt14表达的可能机制。研究方法:1.选用小鼠来源的巨噬细胞株RAW264.7;2.将巨噬细胞接种至细胞培养板,待细胞稳定并且贴壁,将细胞分为12组。正常对照组,H_2O_2 刺激组(0.25mmol/L),H_2O_2 刺激组(0.5mmol/L),H_2O_2刺激组(0.25mmol/L)+ U0126(20mmol/L),H_2O_2刺激组(0.5mmol/L)+U0126(20mmol/L),U0126 抑制组(20mmol/L),H_2O_2刺激组(0.25mmol/L)+ SB203580(40mmol/L),H_2O_2刺激组(0.5mmol/L)+SB203580(40mmol/L),SB203580 抑制组(40mmol/L),H_2O_2 刺激组(0.25mmol/L)+SP600125(10mmol/L),H_2O_2刺激组(0.5mmol/L)+SP600125(10mmol/L),SP600125抑制组(10mmol/L)。3.收集各组细胞并提取蛋白;收集各组培养基。应用细胞免疫荧光法、Western Blot技术分别检测各组细胞中Angptl4的表达;应用ELISA法检测各组培养基中Angpt4的水平;应用Western Blot技术检测MAPKs通路磷酸化蛋白的表达量。实验结果:1.H_2O_2促进巨噬细胞RAW264.7中Angptl4的表达:研究发现不同浓度(0.25mmol/L、0.5mmol/L)的 H_2O_2刺激 RAW264.7 细胞 24 小时,可以促进巨噬细胞中Angpt14的表达,并且呈浓度依赖性(P0.05)。2.H_2O_2激活巨噬细胞RAW264.7中MAPKs通路:研究发现给予RAW264.7细胞 H_2O_2(0.25mmol/L、0.5mmol/L)刺激 24h,细胞中的磷酸化 ERKl/2,p38 MAPK以及JNK通路蛋白表达均增加,且呈浓度依赖性(P0.05)。3.ERK1/2,p38MAPK通路拮抗剂抑制H_2O_2对Angpt14表达的影响:分别在U0126(20mmol/L)和 SB203580(40mmol/L)预处理 90 分钟条件下,H_2O_2(0.25mmol/L、0.5mmol/L)+ U0126(20mmol/L)组和 H_2O_2(0.25mmol/L、0.5mmol/L)+ SB203580(20mmol/L)组中的 Angptl4 的表达量均较单纯H_2O_2刺激组(0.25mmol/L、0.5mmol/L)组明显减少(P0.05),而在 SP600125(lOmmol/L)预处理 30 分钟条件下,H_2O_2(0.25mmol/L、0.5mmol/L)+SP600125(10mmol/L)组和 H_2O_2刺激组(0.25mmol/L、0.5mmol/L)组相比无明显统计学差异(P0.05)。结论及意义:1.H_2O_2可促进巨噬细胞中Angpt14的表达,并呈浓度依赖性。2.H_2O_2可激活巨噬细胞中的MAPKs通路,其中ERK1/2和p38 MAPK通路在H_2O_2调控的Angpt14表达中发挥关键作用。
[Abstract]:Since its discovery, the role of angiopoietin-like protein (4(Angiopoietin-like protein _ 4 / pAngtpl4) in disease has received more and more attention. Studies have shown that Angpt14 is involved in the development of atherosclerosis. However, the exact mechanism of its action is still unclear. Similarly, a large number of studies have shown that oxidative stress is closely related to cardiovascular disease, but the ways in which it works need to be further explored. With the development of the research, the role of oxidative stress and inflammation in atherosclerosis has been revealed further, which has become the focus of research on the pathogenesis of atherosclerosis and the search for new treatment methods. Objective: to investigate the effect of hydrogen peroxide-H _ tid _ 2O _ 2 (H _ 2O _ 2) on the level of angiopoietin like protein (4Angptl4) in mouse macrophage cell line RAW264.7 and the possible mechanism involved in the regulation of Angpt14 expression by H_2O_2. Research method: 1. Mouse macrophage cell line RAW264.7 was selected. The macrophages were inoculated into the cell culture plate and the cells were stable and adhered to the wall. The cells were divided into 12 groups. 姝ｅ父瀵圭収缁，

本文编号：1981794