shRNA特异性静默大鼠肺泡巨噬细胞TLR2基因对染尘大鼠肺纤维化的抑制作用  

Inhibition effect of shRNA specific TLR2 gene silence in rat alveolar macrophage on pulmonary fibrosis in silicosis rats

作　　者：周韶炜[1] 邵伯棕[2] 陈宣辰[2] 张意[3] 

机构地区：[1]同济大学附属上海市肺科医院,上海200433 [2]第二军医大学研管大队临床十队,上海200433 [3]第二军医大学免疫学教研室,上海200433

出　　处：《中国工业医学杂志》2013年第2期88-91,101,共5页Chinese Journal of Industrial Medicine

摘　　要：目的构建表达大鼠TLR2的shRNA慢病毒载体,并研究其在染尘大鼠肺组织内对巨噬细胞功能的抑制效应。方法设计并构建具有干扰效力的shRNA表达质粒,筛选出对TLR2干扰效果最好的一段序列,使用Gateway方法重组到慢病毒表达载体中进行包装,采用包装好的慢病毒感染大鼠肺泡巨噬细胞系NR8383,ELISA法检测TGF-β1、IL-1β、IL-6的表达情况;而后,在大鼠矽肺模型中引入TLR2慢病毒载体,观察矽肺发生和进展情况。结果成功筛选出具有良好TLR2表达干扰效果的shRNA,并成功包装入慢病毒,慢病毒滴度为1.0×106TU/ml,转染慢病毒的肺泡巨噬细胞释放TGF-β1、IL-1β、IL-6均显著减少(P<0.05);转导了干扰病毒载体的矽肺大鼠,其矽肺发生发展受抑情况均优于对照组。结论成功构建了表达大鼠TLR2 shRNA的慢病毒,具有良好的抑制TGF-β1、IL-1β、IL-6功能,并能有效抑制矽肺的发生发展,揭示TLR2可能成为治疗肺纤维化的潜在靶点。Objective To construct the shRNA lentivirus vector expressed rat TLR2 shRNA, and study its inhibitory effect on alveolar macrophage in silica-exposed rats. Methods Design and construct three shRNA expression plasmids with silencing effect, then to screen one with best disturbance effect to TLR2 and packed into lentivirus vector which was used to infect rat alveolar macrophage （NR8383） , detect its expression levels of TGF-β1, IL-1β, IL-6 with ELISA technique. Then alveolar macrophage infected lentivirus vector was introduced to the silica-exposed rats and observe the occurrence and development of silicosis. Results The results showed that the best shRNA was successfully screened and was correctly inserted into the lentivirus, titer of the recombinant lentivirus was 8. 0 × 10^6 TU/ml, and the expressions of TGF-β1, IL-1β and IL-6 in alveolar macrophage were all greatly reduced after virus infection （ P 〈 0. 01 ） ; the inhibition situation of silicosis in the rats infected recombinant lentivirus was obviously prior to the control rats. Conclusions The results showed that the construction of recombinant lentivirus expressed rat TLR2 shRNA was successful, which had favourable inhibitory effect on expression of TGF-β1, IL-1β, IL-6, and could protect the progress of silicosis, which suggested that TLR2 might be a novel potential target for blocking pulmonary fibrosis.

关 键 词：矽肺 Toll样受体2(TLR2) 短发夹RNA(shRNA) 肺泡巨噬细胞 转化生长因子β1(TGF—β1) 白 介素-1β(IL-1β) 白介素6(IL-6) 

分 类 号：R994.3[医药卫生—毒理学]