摘要
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 small interfering RNA（siRNA） expression plasmids of plasminogen activator inhibitor-1 （PAI-1） gene and explore the functions of PAI-1 in pathogenesis of silicotic pulmonary fibrosis. Methods PAI-1-siRNA expressing plasmid was constructed and stably transfected into rat lung fibroblast （FB） by RNA interfering （RNAi） technology. The siRNA with best inhibitory effect was screened. The experiment was divided into three groups. They were control group（C）, in which AM supernatant of（C） not stimulated with SiO2 particulate was added; SiO2 stimulation group（S）, in which AM supernatant of （S） group stimulated with SiO2 particulate was added; and transfection group（T）, in which transfected FB with best inhibitory effect was added first, followed by adding AM supernatant stimulated with SiO2 particulate. The expression of the collagen type I and type III were detected with immunocytochemistry. Results When compared（S）with（C）, the expression of collagen type I and type III in FB increased （P〈0. 05 or P〈0. 01）. When compared（T） with（S）, the expression of collagen type I and type 1]I in FB lowered obviously （P〈 0.01）. Conclusions By RNAi technology PAI-I-siRNA expressing plasmid transfected into FB can make the PAI-1 gene keep silent, and can reduce the extracellular matrix expression. It suggests that PAI-1 plays an important role in the course of silieotic pulmonary fibrosis.
出处
《工业卫生与职业病》
CAS
CSCD
北大核心
2012年第6期327-332,共6页
Industrial Health and Occupational Diseases
基金
河北省自然科学基金资助项目(C2008001007)
河北省科学技术研究与发展计划项目(09276199D)
