魏丛慧, 王瑶, 王凯, 王和静, 张庆锋, 刘志宏. 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[J]. 环境与职业医学, 2016, 33(8): 742-748. DOI: 10.13213/j.cnki.jeom.2016.16327
引用本文: 魏丛慧, 王瑶, 王凯, 王和静, 张庆锋, 刘志宏. 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[J]. 环境与职业医学, 2016, 33(8): 742-748. DOI: 10.13213/j.cnki.jeom.2016.16327
WEI Cong-hui, WANG Yao, WANG Kai, WANG He-jing, ZHANG Qing-feng, LIU Zhi-hong. DNA Methylation Changes of Thymus Gland Differentiation Antigen-1 in Human Embryonic Lung Fibroblast Fibrosis Induced by Coal Dust[J]. Journal of Environmental and Occupational Medicine, 2016, 33(8): 742-748. DOI: 10.13213/j.cnki.jeom.2016.16327
Citation: WEI Cong-hui, WANG Yao, WANG Kai, WANG He-jing, ZHANG Qing-feng, LIU Zhi-hong. DNA Methylation Changes of Thymus Gland Differentiation Antigen-1 in Human Embryonic Lung Fibroblast Fibrosis Induced by Coal Dust[J]. Journal of Environmental and Occupational Medicine, 2016, 33(8): 742-748. DOI: 10.13213/j.cnki.jeom.2016.16327

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

  • 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100 μg/mL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 mRNA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/mL vs(2.498±0.456)ng/mL,(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 mRNA表达(3.38±0.37,3.87±0.28,4.40±0.10)、胶原蛋白Ⅲ mRNA表达(1.65±0.12,2.37±0.19,2.66±0.28)、α-SMA mRNA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-1mRNA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 mRNA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发生甲基化,Thy-1mRNA表达降低,可能是煤尘致肺纤维化发生的重要表观遗传机制之一。

     

    Abstract: 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 500 nmol/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-β1 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 of the 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 timeeffect 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 negative correlation(r=-0.945) with the increasing stimulating time of supernatant fluid(0.634±0.014, 0.448±0.055, 0.352±0.044)(P < 0.05). The mRNA expression levels of DNMTs were significantly higher(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), demonstrating a time-effect relationship with r=0.924, 0.890, and 0.937(P < 0.05). Thy-1 DNA methylation levels were increased in the coal dust groups(0.506±0.014, 0.536±0.017, 0.570±0.032), and also with a time-effect relationship(r=0.682, P < 0.05).

    Conclusion

    Human embryonic lung fibroblasts fibrosis is caused by human monocyte-derived macrophages exposed to coal dust. Thy-1 DNA methylation and decreased Thy-1 mRNA expression are identified in this process, which might be one of the important mechanism of epigenetics in lung fibrosis induced by coal dust.

     

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