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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 shRNA lentivirus vector expressing rat TLR2 and study its inhibitory effect on the function of macrophages in the lung of infected rats. Methods shRNA expression plasmids with interfering potency were designed and constructed. A sequence with the best interference to TLR2 was screened out. The recombinant plasmid was recombined with lentiviral vector by Gateway method and packaged in lentiviral vector. The encapsulated lentivirus infected rat alveolar macrophages The expression of TGF-β1, IL-1β and IL-6 was detected by ELISA. Then TLR2 lentiviral vector was introduced into silicosis model of rat to observe the occurrence and progress of silicosis. Results The shRNA with good TLR2 expression interference was successfully screened and successfully packaged into lentivirus. The titer of lentivirus was 1.0 × 106TU / ml. The lentiviral alveolar macrophages transfected TGF-β1, IL-1β, IL -6 were significantly decreased (P <0.05). Silicosis rats transduced with interfering virus vector showed better development of silicosis than control group. Conclusions The lentivirus expressing rat TLR2 shRNA was successfully constructed and its function of inhibiting the expression of TGF-β1, IL-1β and IL-6 was effectively inhibited and the occurrence and development of silicosis was effectively inhibited. TLR2 may be a potential target for the treatment of pulmonary fibrosis point.