【摘要】 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。更多还原

【Abstract】 Objective To investigate the mechanism of silicosis by observing the effects of silica on the expression of plasminogen activator inhibitor-1（PAI-1）and activator protein-1（AP-1） in human alveolar epithelial cells type Ⅱ（A549）. Methods A549 cell and SiO₂（200 μg /ml） were co-cultured for 0,4,8,16 and 24 h respectively.The reverse transcriptase polymerase chain reaction（RT-PCR）,Western blotting and SP immunocytochemistry were used for detections of the PAI-1 mRNA and protein expression.The nucleoprotein and total protein expression of AP-1 were investigated by Western blotting. Results The expression levels of PAI-1 mRNA and protein were increased in a time-dependent manner（r mRNA=0.911,r protein=0.902,P<0.05）.The expressions of PAI-1 mRNA and protein in experimental groups were higher than that in control group（P<0.05）and was the highest in 24 h group[（0.73±0.01）vs（0.36±.03）].The nucleoprotein expressions of c-jun/c-fos in experimental groups were also higher than those in control group（P<0.05）,and the nucleoprotein expression level of c-jun was the highest in 4 h group[（1.54±0.02）vs（0.56±0.03）];the nucleoprotein expression level of c-fos was the highest in 8 h group[（0.36±0.01）vs（0.15±0.01）].Both c-jun and c-fos expression were decreased after 16 h,but the total protein expressionof c-jun/c-fos had no difference in all experimental groups.The positive signal of PAI-1 was located in cytoplasm and nucleus. Conclusion SiO₂ could induce PAI-1 expression of A549 in a time-dependent manner,and AP-1 activation can be observed in early time.更多还原