巨噬细胞移动抑制因子经Rho激酶途径促进人胚肺成纤维细胞表型转化

摘要

目的:观察重组巨噬细胞移动抑制因子(rMIF)对人胚肺成纤维细胞(MRC-5)表型转换的作用,探讨哮喘气道重塑的发生机制.方法:不同浓度的rMIF (25～100 μg/L)分别作用于MRC-5细胞24 h或48 h,RT-PCR法检测α-平滑肌肌动蛋白(α-SMA) mRNA表达,Western blotting检测α-SMA蛋白合成;100 μg/L rMIF刺激MRC-5细胞48 h,刺激前0.5 h加入Rho拮抗剂Y27632,RT-PCR法检测α-SMA mRNA表达,Western blotting法检测α-SMA表达;100 μg/L rMIF分别刺激MRC-5 6 h、12 h、24 h或48 h,Western blotting检测磷酸化肌球蛋白磷酸酶目标亚单位1(p-MYP1)蛋白合成.结果:刺激MRC-5 24 h,不同浓度的rMIF对α-SMA mRNA转录未见显著影响(P>0.05).刺激MRC-5细胞48 h,rMIF可呈剂量依赖地促进α-SMA mRNA(r=0.697,P<0.01)和蛋白(r=0.957,P<0.01)表达.Y27632预刺激后,显著抑制rMIF100 μg/L促进α-SMA mRNA和蛋白表达的作用(均P<0.01).rMIF刺激6 h后,p-MYPT1表达显著增加(P< 0.01),12 h达到高峰(P<0.01),24 h开始下降,但24 h和48 h其水平仍高于对照组(均P<0.01).结论:rMIF通过Rho信号通路刺激成纤维细胞表型转化,可能在哮喘气道重塑的发病机制中发挥关键作用.%AIM: To investigate the influence and the mechanism of recombinant macrophage migration - in-hibitory factor (rMIF) on fibroblasts. METHODS: Cultured human embryonic lung fibroblasts ( MRC -5 cells) were di-vided into 2 groups: the cells in treatment group were treated with rMIF (25～100 μg/L) for 24 h or 48 h, and the control cells were without rMIF treatment. The mRNA expression of a - SMA was examined by RT - PCR. The protein level of arn- SMA induced by rMIF was quantified by Western blotting. Half an hour before 100 μg/L rMIF challenge, Y27632 was added to the cells of 2 groups. After challenged for 48 h, the total RNA and protein were extracted, and the expression of arn- SMA at mRNA and protein levels was determined by means of RT - PCR and Western blotting. After challenged by 100 μg/L rMIF for 6 h, 12 h, 24 h and 48 h, the cell total protein was extracted, and the protein level of a - SMA induced by rMIF was quantified by Western blotting. RESULTS: After stimulation for 24 h, rMIF did not increase the a - SMA mR-NA synthesis compared with control. After stimulation for 48 h, rMIF significantly increased the expression of a - SMA mRNA and protein in a dose - dependent manner compared with control ( r = 0. 697 and r = 0. 957, both P < 0. 01 ) .rnY27632 pretreatment prevented the increase ( P < 0. 01) . The amount of phosphorylated MYPT1 increased at 6 h ( P < 0. 01) , reached the maximum at 12 h ( P < 0. 01) , and decreased but still higher than the control at 24 h and 48 h ( P < 0. 01). CONCLUSION: rMIF increases the α - SMA synthesis in MRC -5 cells and Rho - kinase regulates this process, suggesting that MIF may play important roles in the pathogenesis of airway remodeling.