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[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发生甲基化,Thy-1m RNA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。
[Objective] To observe the expression of Thy-1 and its DNA methylation during the process of MRC-5 fibrosis induced by coal dust in monocyte-derived macrophages. [Methods] Human monocytes were cultured in 1640 medium containing 500 nmol / L phorbol ester and induced to differentiate into macrophages after 24 h. The culture supernatants of macrophages stimulated with 100 μg / m L of coal dust for 24 h were collected, Restimulation of MRC-5 24,48,72 h, the control group were normal macrophages and normal human embryonic lung fibroblasts. The levels of IL-6 and TGF-β1 in macrophage supernatant and the expression of TGF-β1 in MRC-5 were detected by enzyme linked immunosorbent assay (ELISA), and the expressions of collagen-I, collagen- , Α-SMA, Thy-1, DNMTs, 1,3a and 3b m RNA were detected by nested PCR. Thy-1 DNA methylation was detected by nested-specific PCR. [Results] The levels of IL-6 and TGF-β1 in the supernatant of macrophage in the coal dust group were significantly increased compared with the control group [(5.542 ± 0.953) ng / m Lvs (2.498 ± 0.456) ng / m L, (462.435 (P <0.05). The expression of TGF-β1 protein in MRC-5 cells was up-regulated after macrophages were stained with dust [(223.813 ± 0.56 ± 2.62) ng / L vs 329.971 ± 17.911 ng / L 5.723), (263.757 ± 17.254), (326.720 ± 11.263) ng / L and collagen I m RNA expression (3.38 ± 0.37, 3.87 ± 0.28, 4.40 ± 0.10, 2.37 ± 0.19 and 2.66 ± 0.28 respectively), the expression of α-SMA m RNA (2.41 ± 0.47,4.76 ± 0.10,4.23 ± 0.63) was increased, and there was a time-dependent relationship between them. The r values were 0.965,0.876,0.899,0.667 (P <0.05). The expression of Thy-1mRNA in the MRC-5 coal dust group was lower than that in the control group. The expression of Thy-1mRNA in MRC-5 group decreased gradually with the increase of macrophage supernatant (0.634 ± 0.014,0.448 ± 0.055,0.352 ± 0.044) (r = -0.945, P <0.05). The DNMTs mRNA expression in MRC-5 group was higher than that in control group (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), and there are time-effect relationship, r score (0.506 ± 0.014,0.536 ± 0.017,0.570 ± 0.032) in the coal dust group, and there was a time-effect relationship (r = 0.682 , P <0.05). [Conclusion] Coal dust could induce fibroblast fibrosis in human embryo fibroblasts by monocyte-derived macrophages. Thy-1 DNA methylation and Thy-1m RNA expression decreased during the process, which may be caused by coal dust One of the important epigenetic mechanisms of pulmonary fibrosis.