慢病毒介导shRNA特异性沉默TLR4基因对大鼠肺泡巨噬细胞功能的影响

Effect of lentivirus expressing TLR4 shRNA on function of rat alveolar macrophage

目的构建表达大鼠TLR4 shRNA的慢病毒,并观察其对大鼠肺泡巨噬细胞TLR4的抑制作用和对内毒素(LPS)诱导的IL-6、IL-1β释放的影响。方法设计并构建4个可能具有干扰效力的shRNA表达质粒,将他们分别与预先构建好的TLR4表达质粒共转染HEK-293T细胞,筛选出一段干扰效果最好的shRNA,使用Gateway的方法重组到慢病毒表达载体中并进行病毒包装和滴度测定,使用包装好的慢病毒感染大鼠肺泡巨噬细胞系NR8383并加入LPS刺激,ELISA检测IL-1β、IL-6的释放情况。结果成功筛选出具有较高干扰效率的shRNA,并成功包装入慢病毒,慢病毒滴度为2.0×106TU/ml。转染慢病毒后的LPS诱导的肺泡巨噬细胞IL-1β、IL-6的释放均明显减少(P<0.05)。结论成功构建了表达大鼠TLR4 shRNA的慢病毒,具有良好的抑制IL-1β、IL-6表达的作用,为下一步动物体内实验基因治疗大鼠肺移植后的慢性排斥反应奠定良好的基础。

Objective To construct the replication-incompetent lentivirus expressing rat TLR4 shRNA,and to observe its inhibitory effect on TLR4 expression in alveolar macrophage and on lipopolysaccharide （LPS）-induced release of IL-1β,IL-6.Methods We designed and constructed four shRNA expressing plasmids with silencing effect,then they were co-transfected into HEK293T cells with the TLR4 eukaryotic vector constructed previously;the best shRNA was selected to be packed into lentivirus;and the titer was determined.The packed lentvirus was used to infect the alveolar macrophage（NR8383） in presence of LPS,and the LPS-induced IL1β,IL-6 expression was examined by ELISA.Results The best shRNA was successfully screened out and was correctly inserted into the lentivirus.The titer of the recombinant lentivirus was 2.0 × 106 TU/ml.The LPS-induced expression of IL-1β,IL-6 in the alveolar macrophages was greatly reduced after virus infection （P〈0.05）.Conclusion We have successfully constructed the recombinant lentivirus expressing rat TLR4 shRNA,which has an satisfactory inhibitory effect against expression of IL-1β,IL-6,paving a way for studying the chronic rejection reaction after rat lung transplantion in vivo.