纤维溶解酶原激活物抑制剂-1对大鼠胚肺成纤维细胞的影响

The effect of plasminogen activator inhibitor-1 on embryonic lung fibroblasts from rats

目的 研究纤维溶解酶原激活物抑制剂-1（PAI-1）对大鼠胚肺成纤维细胞增殖、转化及胶原合成的影响,探讨PAI-1在肺纤维化发生中的作用.方法 体外分离培养Wister 待产孕大鼠胚肺成纤维细胞,应用四甲基偶氮唑盐试验观察不同浓度（5、10、20、40、80 μg/L）的PAI-1刺激12、24和48 h后成纤维细胞的增殖率;选用PAI-1最适浓度20 μg/L刺激48和72 h后,细胞免疫化学法检测增殖细胞核抗原（PCNA）的表达;实时PCR法检测α-平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原24和48 h时mRNA表达的变化.结果各浓度PAI-1刺激成纤维细胞后,以20 μg/L 12 h增殖率及吸光度值最高,分别为62.6%和0.573±0.039.在12 h中,不同浓度组较对照组差异有统计学意义（F=111.112,P=0.000）,选择20 μg/L为最适浓度.免疫细胞化学法检测20 μg/L PAI-1刺激48、72 h后,PCNA表达的积分吸光度值分别为3685±686和2530±477,较对照组差异有统计学意义（F=7.85,P=0.020）.PAI-1刺激成纤维细胞24和48 h后,α-SMA表达上调（230±11）%和（159±9）%,较对照组差异有统计学意义（F=39.92,P=0.000）.Ⅰ型胶原表达上调（92±8）%和（65±12）%,差异有统计学意义（F=32.61,P=0.001）.结论 PAI-1可以促进成纤维细胞增殖、转化及胶原合成,可能促进肺间质纤维化的发生和发展.

Objective To explore the effect of plasminogen activator inhibitor-1 （PAI-1） on the proliferation and conversion of rat embryonic lung fibroblasts and the synthesis of collagen, and therefore to explore the function of PAI-1 in pulmonary fibrosis.Methods The embryonic lung fibroblasts from pregnant Wistar rats were isolated and cultured in vitro. The reproduction rate of fibroblasts at 12 h, 24 h, and 48 h after being stimulated by PAI-1 with different concentrations （5, 10, 20, 40, 80, and 100 μg/L） was measured by MTT assay. After being stimulated by PAI-1 with the most suitable concentration （20 μg/L） for 48 h and 72 h, the expression of proliferating cell nuclear antigen （PCNA） was measured by immunocytochemical technique, and the mRNA expression of α-SMA and type-1 collagen at 24 h and 48 h was measured by real-time PCR. Results PAI-1 with different concentrations stimulated the proliferation of fibroblasts. The highest proliferation rate and absorbance in concentration of 20 μg/L and at 12 h were 62.6% and 0.573±0.039. The comparison of different concentrations showed that the difference was significant（F=111.112,P=0.000）. Therefore, 20 μg/L was selected as the most suitable concentration. Using immunocytochemical method, the optical density of PCNA at 48 h and 72 h were 3685±686 and 2530±477 after being stimulated with 20 μg/L PAI-1.The comparison showed significant difference（F=7.85,P=0.02）. The expression of α-SMA increased （230±11）% and（159±9）% at 24 h and 48 h after being stimulated with 20 μg/L PAI-1, and the difference was significant（F=39.92, P=0.0003）. The expression of type-1 collagen increased（92±8）% and （65±12）%, the difference being significant（F=32.61,P=0.0006）. Conclusion PAI-1 can promote the proliferation and conversion of fibroblasts and the synthesis of collagen, which may be involved in the pathogenesis of pulmonary interstitial fibrosis.