摘要
目的通过观察硫酸铍刺激人胚肺成纤维细胞(MRC-5细胞)所致胸腺细胞分化抗原-1(Thy-1)DNA甲基化改变情况,探讨Thy-1在硫酸铍致肺纤维化中的作用。方法建立离体MRC-5细胞培养实验模型,以终浓度分别为1.0、10.0和100.0μmol/L的硫酸铍染毒(设为低、中、高剂量组),对照组不作染毒处理;另设甲基化转移酶抑制剂5-氮杂胞苷(AZC)干预组(予终浓度为10.0μmol/L硫酸铍和10.0μmol/L AZC处理)和组蛋白去乙酰化酶抑制剂曲古抑菌素A(TSA)干预组(予终浓度为10.0μmol/L硫酸铍和0.5μmol/L TSA处理)。分别在染毒24、48和72 h后收集细胞,采用实时定量式聚合酶链式反应检测胶原蛋白Ⅰ、胶原蛋白Ⅲ和α-平滑肌肌动蛋白(α-SMA)和Thy-1的mRNA相对表达水平,巢式降落式甲基化特异性聚合酶链式反应检测Thy-1 DNA甲基化水平。结果在24 h时间点,MRC-5细胞中胶原蛋白ⅢmRNA相对表达水平呈随染毒剂量增加而增加的趋势(P<0.05);在48和72 h时间点,MRC-5细胞中胶原蛋白Ⅰ、胶原蛋白Ⅲ和α-SMA的mRNA相对表达水平均呈随染毒剂量增加而增加的趋势(P<0.05);3个剂量组MRC-5细胞上述3个指标均呈随着染毒时间的增加而增加趋势(P<0.05)。3个剂量组MRC-5细胞Thy-1 mRNA相对表达水平均低于对照组(P<0.05),高剂量组MRC-5细胞Thy-1 mRNA相对表达水平低于低剂量组(P<0.05)。中、高剂量组Thy-1 DNA甲基化水平均高于对照组(P<0.05),3个剂量组Thy-1 DNA甲基化水平呈随染毒剂量增加而增加趋势(P<0.05)。2个干预组MRC-5细胞Thy-1 DNA甲基化水平均高于对照组(P<0.05),但与中剂量组差异均无统计学意义(P>0.05)。结论硫酸铍刺激可导致MRC-5细胞纤维化的发生;该过程中Thy-1 DNA甲基化程度升高,Thy-1 mRNA表达水平降低,可能是硫酸铍致肺纤维化的重要机制之一。
Objective The change of DNA methylation of thymocyte differentiation antigen-1（ Thy-1） was observed in beryllium sulfate（ Be SO4） stimulated human fetal lung fibroblast（ MRC-5 cell） to explore the effects of Thy-1 in Be SO4 induced lung fibrosis. Methods MRC-5 cell culture in vitro model was used. The final concentrations of Be SO4were1. 0,10. 0 and 100. 0 μmol / L（ low-,medium- and high-dose groups）. The control was untreated. Other 2 intervention groups were the 5-azacytidine（ AZC） intervention group（ 10. 0 μmol / L of AZC and 10. 0 μmol / L Be SO4） and the trichostatin A（ TSA） intervention group（ 0. 5 μmol / L of TSA and 10. 0 μmol / L Be SO4）. The cells were collected 24,48 and 72 hours after exposure. Real-time quantitative polymerase chain reaction（ PCR） was used to determine the relative expression of collagen typeⅠ（ Col Ⅰ）,collagen type Ⅲ（ Col Ⅲ）,α-smooth muscle actin（ α-SMA） and Thy-1 mRNA.The nested landed methylation specific PCR was used to detect the Thy-1 DNA methylation level. Results At 24 hours,the relative expression level of Col Ⅲ mRNA in MRC-5 cells showed an increasing trend with increasing dose（ P〈0. 05）;at 48 and 72 hours,the relative expression levels of Col Ⅰ,Col Ⅲ and α-SMA mRNA in MRC-5 cells increased with the increasing dose（ P〈0. 05）. All these 3 indicators in MRC-5 cells of 3 dose groups increased with the increase of expose time（ P〈0. 05）. The relative expression level of Thy-1 mRNA in MRC-5 cells of all 3 dose groups were lower than that in control（ P〈0. 05）. The relative expression level of Thy-1 mRNA of the high-dose group was lower than that of the lowdose group（ P〈0. 05）. The Thy-1 DNA methylation levels in the medium- and high-dose groups were both higher than that of the control（ P〈0. 05）. The Thy-1 DNA methylation levels of the 3 dose groups increased with the increasing dose（ P〈0. 05）. The Thy-1 DNA methylation levels of MRC-5 cells in the 2 intervention groups were higher than that of the control（ P〈0. 05）,but there was no significant difference when compared with the medium-dose group（ P〈0. 05）.Conclusion Be SO4 stimulation can induce the fibrosis of MRC-5 cells. In this process,the Thy-1 DNA methylation level increases,while the Thy-1 mRNA expression level decrease. Thy-1 DNA methylation might be one of the important mechanisms of lung fibrosis induced by Be SO4.
出处
《中国职业医学》
CAS
北大核心
2016年第1期20-25,共6页
China Occupational Medicine
基金
国家自然科学基金(81360415)
