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目的研究SiO2对人肺泡Ⅱ型上皮细胞(A549)中纤溶酶原激活物抑制因子1(PAI1)及激活蛋白1(AP1)表达的影响,探讨SiO2致肺纤维化的发病机制。方法按SiO2(200μg/ml)与A549细胞共育时间的不同,实验分SiO2刺激0、4、8、16、24h组。利用逆转录聚合酶链反应(RTPCR)、Westernblot及免疫细胞化学SP法检测细胞PAI1mRNA和蛋白的表达;AP1(cjun/cfos)总蛋白及核蛋白表达的检测采用Westernblotting。结果SiO2刺激后,实验组PAI1mRNA及蛋白表达强度随时间延长依次增强,呈直线正相关(rmRNA=0.911,r蛋白=0.902,P<0.05),且实验组PAI1蛋白表达均高于对照组(0.36±0.03),其中24h组(0.73±0.01)表达最强;cjun和cfos核蛋白的表达亦明显高于对照组(0.52±0.02、0.15±0.01),cjun核蛋白的表达4h组(1.54±0.02)最强;cfos核蛋白表达8h组(0.36±0.01)最强;cjun和cfos核蛋白的表达均从孵育16h开始逐渐下降。AP1(cjun/cfos)总蛋白的表达各组间差异无统计学意义(P>0.05);PAI1阳性信号定位于胞浆和胞核。结论SiO2能够诱导A549细胞PAI1的表达,呈现出明显的时间-效应关系,核转录因子AP1在SiO2刺激的早期被激活。
Objective To investigate the effect of SiO2 on the expression of plasminogen activator inhibitor 1 (PAI1) and activator protein 1 (AP1) in human alveolar type Ⅱ epithelial cells (A549) and to explore the pathogenesis of SiO2-induced pulmonary fibrosis. Methods According to the difference of co-incubation time between SiO2 (200μg / ml) and A549 cells, the rats were divided into 0, 4, 8, 16 and 24 hours groups. The expression of PAI1 mRNA and protein was detected by reverse transcription polymerase chain reaction (RTPCR), Western blot and immunocytochemistry (SP). The total protein and nuclear protein expressions of AP1 (cjun / cfos) were detected by Western blotting. Results After stimulated with SiO2, the expression of PAI1 mRNA and protein in the experimental group increased with time and increased linearly (rmRNA = 0.911, r = 0.902, P <0.05), and the expression of PAI1 protein in the experimental group was significantly higher than that in the control group ± 0.03). The expression of nuclear cjun and cfos protein was also significantly higher than that of the control group (0.52 ± 0.02,0.15 ± 0.01), cjun nuclear protein expression of 4h group (1.54 ± 0.02) ) Was the strongest; the expression of cfos nucleoprotein was the strongest in 8h group (0.36 ± 0.01); the expression of cjun and cfos nucleoprotein gradually decreased from 16h after incubation. The expression of AP1 (cjun / cfos) protein in all groups had no significant difference (P> 0.05). PAI1 positive signal located in cytoplasm and nucleus. Conclusion SiO2 can induce the PAI1 expression in A549 cells, showing a significant time-effect relationship. The nuclear transcription factor AP1 is activated in the early stage of SiO2 stimulation.