煤尘致人胚肺成纤维细胞纤维化中胸腺分化抗原-1 DNA甲基化的变化  

DNA Methylation Changes of Thymus Gland Differentiation Antigen-1 in Human Embryonic Lung Fibroblast Fibrosis Induced by Coal Dust

作　　者：魏丛慧 王瑶[1] 王凯[1] 王和静 张庆锋[2] 刘志宏[1] 

机构地区：[1]宁夏医科大学公共卫生与管理学院,宁夏银川750004 [2]辽宁省疾病预防控制中心业务与应急管理办公室,辽宁沈阳110000

出　　处：《环境与职业医学》2016年第8期742-748,共7页Journal of Environmental and Occupational Medicine

基　　金：2013年宁夏自治区科技支撑计划项目(DNA甲基化抑制剂在职业性肺纤维化防治中的作用研究;无编号)

摘　　要：[目的]观察煤尘通过单核细胞源性巨噬细胞致人胚肺成纤维细胞MRC-5纤维化改变过程中,胸腺分化抗原-1(Thy-1)表达及其DNA甲基化变化。[方法]用含500 nmol/L佛波酯的1640培养液培养人单核细胞,24 h诱导分化后成巨噬细胞;收集100μg/m L煤尘刺激巨噬细胞24 h的培养上清,再刺激MRC-5 24、48、72 h,对照组分别为正常巨噬细胞和正常人胚肺成纤维细胞。酶联免疫分析法测定巨噬细胞上清液中IL-6、TGF-β1蛋白表达水平和MRC-5中TGF-β1蛋白表达水平,实时荧光定量PCR法检测胶原蛋白-I、胶原蛋白-III、α-SMA、Thy-1、DNA甲基化转移酶(DNMTs)1、3a、3b m RNA的表达,巢式降落式特异性PCR检测Thy-1DNA甲基化。[结果]煤尘组巨噬细胞上清中IL-6、TGF-β1蛋白表达水平与对照组比较明显增加[(5.542±0.953)ng/m Lvs(2.498±0.456)ng/m L,(462.435±56.620)ng/L vs(329.971±17.911)ng/L](P<0.05);巨噬细胞染尘后上清刺激MRC-5 24、48、72 h,TGF-β1蛋白表达[(223.813±5.723),(263.757±17.254),(326.720±11.263)ng/L]和胶原蛋白I m RNA表达(3.38±0.37,3.87±0.28,4.40±0.10)、胶原蛋白III m RNA表达(1.65±0.12,2.37±0.19,2.66±0.28)、α-SMA m RNA表达(2.41±0.47,4.76±0.10,4.23±0.63)均升高;且均存在时间效应关系,r值分别为0.965,0.876,0.899,0.667(P<0.05);煤尘组MRC-5中Thy-1m RNA表达均低于对照组,随着巨噬上清液作用时间的增加,其表达逐渐降低(0.634±0.014,0.448±0.055,0.352±0.044),呈负相关(r=-0.945,P<0.05);煤尘组MRC-5中DNMTs m RNA表达均较对照组升高(DNMT1:1.57±0.12,2.51±0.33,7.00±0.71;DNMT3a:0.74±0.09,2.89±0.52,3.31±0.53;DNMT3b:1.46±0.18,2.25±0.44,5.33±0.16),且均存在时间效应关系,r值分别为0.924,0.890,0.937(P<0.05);煤尘组Thy-1 DNA甲基化水平较对照组升高(0.506±0.014,0.536±0.017,0.570±0.032),存在时间效应关系(r=0.682,P<0.05)。[结论]煤尘通过单核细胞源性巨噬细胞致人胚肺成纤维细胞纤维化发生,在此过程中Thy-1 DNA发生甲基�[ Objective ] To observe the changes of thymus gland differentiation antigen-1 （Thy-1） expression and its DNA methylation in human embryonic lung fibroblasts （MRC-5） fibrosis induced by coal dust through human monocytic cell derived macrophages. [ Methods ] We stimulated human monocytes differentiation to macrophages by using 500nmol/L phorbol 12-myristate 13-acetate （PMA） in 1640 medium for 24 h; collected supernatant of 100 μg/mL coal dust stimulated macrophages after 24 h to stimulate human embryonic lung fibroblasts for 24, 48, and 72 h. The control groups were normal macrophages and normal MRC-5. The protein levels of IL-6 and TGF-β1 of macrophages and TGF-IM of MRC-5 in the supernatant were determined by enzyme-linked immunoassay; collagen-Ⅰ, collagen-Ⅲ, α-SMA, Thy-1, and DNA methylation transferase （DNMTs） （1, 3a, 3b） mRNA expression levels were detected by real-time fluorescent quantitative PCR; DNA methylation of Thy-1 was detected by nested touchdown specific PCR. [ Results ] The protein expression levels of IL-6 and TGF-β1 in macrophage supernatant ofthe coal dust groups were significantly increased compared with the control group [（5.542 ± 0.953）ng/mL vs （2.498 ± 0.456）ng/mL, （462.435 ± 56.620）ng/L vs （329.971 ± 17.911）ng/L, respectively] （P 〈 0.05）. After MRC-5 were stimulated with coal dust treated macrophages for 24, 48, and 72h, the TGF-β1 protein expression levels [（223.813 ± 5.723）, （263.757 ± 17.254）, （326.720 ± 11.263） ng/L] as well as collagen-I （3.38 ± 0.37, 3.87 ± 0.28, 4.40± 0.10）, collagen-Ⅲ （1.65 ± 0.12, 2.37 ± 0.19, 2.66 ± 0.28）, and α-SMA （2.41 ± 0.47, 4,76 ± 0.10, 4.23 ± 0.63） mRNA expression levels were significantly higher than those of the control group, and in a time- effect relationship with r=0.965, 0.876, 0.899, and 0.667, respectively （P〈 0.05）. The mRNA expression levels of Thy-1 in MRC-5 of the coal dust groups were all lower than those of the control group, and there was a ne

关 键 词：煤尘 单核细胞源性巨噬细胞 人胚肺成纤维细胞 纤维化 胸腺分化抗原-1 DNA甲基化 

分 类 号：R114[医药卫生—卫生毒理学]