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目的构建纤溶酶原激活物抑制因子-1(PAI-1)基因小干扰RNA片段(siRNA)表达质粒,探讨PAI-1基因在矽肺纤维化发病中的功能。方法利用RNA干扰技术,将构建的PAI-1-siRNA表达质粒转染大鼠肺成纤维细胞(FB),筛选最佳干扰序列。实验分3组:对照组(C),加入未经二氧化硅(SiO2)粉尘刺激的肺泡巨噬细胞(AM)培养上清;SiO2刺激组(S),加入经SiO2粉尘刺激的AM培养上清;转染组(T),先将筛选出最佳干扰序列的质粒转染FB,再加入经SiO2粉尘刺激的AM培养上清。用免疫细胞化学法检测各组I、Ⅲ型胶原蛋白的表达。结果经条件上清刺激后,S组与C组比较,FB中I、Ⅲ型胶原表达增加(P<0.05或P<0.01);T组与S组比较,FB中Ⅰ、Ⅲ型胶原的表达明显下降(P<0.01)。结论通过RNA干扰技术将PAI-1-siRNA表达质粒转染FB,可使PAI-1基因沉默,且可减少细胞外基质的沉积,提示PAI-1基因与矽肺纤维化的发生、发展密切相关。
Objective To construct plasminogen activator inhibitor-1 (PAI-1) small interfering RNA (siRNA) expression plasmids and investigate the function of PAI-1 gene in the pathogenesis of silicotic fibrosis. Methods PAI-1-siRNA expression plasmids were transfected into rat lung fibroblasts (FBs) using RNA interference technique to screen for the best interference sequences. The experiment was divided into three groups: the control group (C), the supernatant of alveolar macrophages (AM) stimulated with SiO2 powder was added; the SiO2 stimulus group (S) In the transfection group (T), the plasmids with the best interference sequences were selected and transfected into FBs. The supernatant of AMs stimulated by SiO2 dusts was added. Immunocytochemistry was used to detect the expression of type I and type III collagen. Results Compared with group C, the expression of type I and type III collagen in FB increased (P <0.05 or P <0.01), and the expression of type I and type III collagen in FB increased significantly Significantly decreased (P <0.01). Conclusions Transfection of PAI-1-siRNA expression plasmids into FBs by RNA interference can silence PAI-1 gene and decrease the deposition of extracellular matrix, suggesting that PAI-1 gene is closely related to the occurrence and development of silicosis fibrosis.