雾霾对小鼠肺巨噬细胞株IL-17、HDAC2水平变化的影响

发布时间：2018-05-14 13:47

本文选题：慢性阻塞性肺疾病 + PM2.5　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:慢性阻塞性肺疾病(Chronic obstructive pulmonary disease,COPD)与慢性炎症有关,主要影响肺实质和外周气道,导致大部分不可逆和渐进的气流受限。这种炎症的特点是肺泡巨噬细胞、中性粒细胞、T淋巴细胞(主要是Th1和Th17细胞亚群)数量增加,和从循环中招募的先天淋巴样细胞[1]。空气动力学当量直径小于或等于2.5μm的颗粒物,也称细颗粒物,也是大气总悬浮颗粒物(Total suspended particulate matter,TSP)的一部分,属于具有重要卫生学意义的空气污染物,可吸附大量的有毒、有害物质,易进入呼吸道深部引发呼吸道损伤。肺巨噬细胞是呼吸道内部重要的防御屏障,能够吞噬外来颗粒物,分解清除外来细菌。,肺巨噬细胞受到损伤,会释放大量细胞因子,增加呼吸系统疾病的发病率和死亡率[2]。COPD造成全球健康问题的增加,其预测和预后的新的生物学标记物是迫切必要的。本实验通过可入肺细颗粒物(PM2.5)刺激小鼠肺巨噬细胞株(RAW264.7细胞),观察白细胞介素17(Interleukin 17,IL-17)及组蛋白去乙酰化酶-2(Histone deacetylase2,HDAC2)水平变化,探讨其在PM2.5致病中的作用,及是否IL-17、HDAC2水平可作为一种很有前途的,很容易检测的生物标志物。方法:1酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)测细胞上清液中IL-17的浓度。首先常规培养传代RAW264.7细胞,调节细胞浓度为5×106/ml,于24孔板中培养过夜,随机分为6组。(1)对照组:加入与实验组等体积的无血清1640培养液,孵育细胞;(2)LPS组:培养液中加入LPS,调整终浓度100ng/ml,孵育细胞;(3)PM2.5 25ug/ml组:培养液中加入终浓度为25ug/ml PM 2.5悬液,孵育细胞;(4)PM2.5 50ug/ml组:培养液中加入终浓度为50ug/ml PM 2.5悬液,孵育细胞;(5)PM2.5 100ug/ml组:培养液中加入终浓度为100ug/ml PM 2.5悬液,孵育细胞;(6)PM2.5200ug/ml组:培养液中加入终浓度为200ug/ml PM 2.5悬液,孵育细胞。各组分别刺激12h后取细胞上清液,-80℃保存,ELISA法检测IL-17含量。2 HDAC2活性比色法定量测定试剂盒检测细胞HDAC2活性。实验仍按上述分组对RAW246.7进行刺激后,比色法定量检测HDAC2活性。结果:1细胞上清液中IL-17含量变化。与阴性对照组比较,LPS刺激RAW264.7细胞后,细胞上清液中IL-17含量增加;PM2.5刺激RAW246.7细胞,IL-17含量亦增加;PM2.5浓度50ug/ml组,IL-17含量最高,PM2.5100ug/ml、200ug/ml组较PM2.5浓度50ug/ml组IL-17含量降低。但比阴性对照组增高,比较差异具有统计学意义(P0.05),PM2.5浓度50ug/ml组与PM2.5浓度100ug/ml组IL-17比较差异无统计学意义。2活性比色法定量测定HDAC2活性变化:与阴性对照组比较,LPS刺激RAW246.7细胞后,细胞HDAC2活性有所降低;PM2.5刺激RAW264.7细胞,细胞HDAC2活性降低,且随PM2.5浓度增高,HDAC2活性显著降低(P0.05)。结论:1 PM2.5不同浓度组刺激分泌IL-17增加,提示IL-17参与了细胞损伤的发生发展过程。PM2.5浓度50ug/ml组100ug/ml组IL-17含量无明显差异,PM2.5浓度200ug/ml组IL-17含量反而降低,说明高浓度PM2.5可能引起细胞存活率降低。2 LPS及PM2.5刺激RAW264.7细胞引起组蛋白乙酰化增强,导致前炎症因子IL-17高表达,促进炎症反应发生。
[Abstract]:Objective : Chronic obstructive pulmonary disease ( COPD ) is associated with chronic inflammation , which mainly affects the pulmonary parenchyma and the peripheral airway , resulting in most irreversible and progressive restriction of the airflow . This inflammation is characterized by an increase in the number of alveolar macrophages , neutrophils , T lymphocytes ( mainly Th1 and Th17 cells ) , and congenital lymphoid cells recruited from the cycle . The air dynamic equivalent diameter is less than or equal to 2.5 . m u.m , also referred to as fine particles , is part of total suspended particulate matter ( TSP ) , and belongs to air pollutants with important hygienic significance , and can adsorb large amounts of toxic and harmful substances , and is easy to enter the deep part of the respiratory tract to cause respiratory tract injury . Methods : The levels of IL - 17 , IL - 17 and Histone defibrase - 2 ( HDAC2 ) were measured by enzyme - linked immunosorbent assay ( ELISA ) . ( 1 ) In the control group , the serum levels of IL - 17 and IL - 17 in the culture medium were determined by adding LPS to the culture solution , the final concentration was 100 ng / ml , the concentration of IL - 17 was increased , the cells were incubated ; ( 4 ) PM2 . 5200ug / ml group : the concentration of IL - 17 was higher than that in the negative control group . Conclusion : The levels of IL - 17 and IL - 17 in PM2.5 stimulated RAW264.7 cells decreased significantly ( P0.05 ) . Conclusion : The levels of IL - 17 in PM2.5 stimulated RAW264.7 cells were not significantly different .

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.9

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