慢病毒介导siRNA干扰MyD88表达对大鼠肺泡巨噬细胞功能的影响

目的采用RNA干扰技术，通过构建表达大鼠髓样分化因子88（MyD88）siRNA慢病毒干扰大鼠肺泡巨噬细胞MyD88g／表达，检测其对细胞功能的影响。方法针对MyD88基因，设计并构建3对siRNA表达质粒，分别与预先构建好表达MyD88N质粒共转染HEK-293T细胞，Westernblot检测MyD88的表达情况，筛选出其中1对干扰效率最高的siRNA，用Gateways方法构建慢病毒干扰载体包装成慢病毒，将慢病毒转染大鼠肺泡巨噬细胞系NR8383胞并加入内毒素（LPS）诱导，未转染慢病毒的细胞作为空白对照组和LPS激活组：空白对照组加入与LPS等体积的PBS；LPS激活组加入LPS刺激。ELISA测定各组细胞因子（IL-18、IL-6）释放情况。结果成功筛选出了1对干扰效率最高的siRNA并包装成慢病毒，病毒滴度为2．0×10^6TU／ml。LPS诱导后，与对照组相比．感染慢病毒的NR8383细胞MyD88表达明显受到抑制，IL-1β、IL-6的释放均显著减少，差异有统计学意义。结论慢病毒介导RNA干扰能够有效抑制NR8383细胞MyD88基因的表达，显著减少胞因子的释放，为以抗原提呈细胞（APC）为靶向的体内实验治疗大鼠肺移植相关闭塞性细支气管炎（obliterative bronchitis，OB）的研究提供了手段。

Impact of lentivirus-mediated siRNA interfering MyD88 on rat alveolar macrophage

Objective To detecte rat alveolar macrophage function after using RNA interference technology, to interfere expression of MyD88 on rat alveolar macrophage by constructing recombinant lentivirus that express rat myeloid differentiation factor 88 （MyD88） siRNA. Methods Designed and constructed 3 plasmid expressing MyD88 siRNA, then respectively co-transfected them into HEK- 293T ceils with plasmid expressing MyD88. Western blotting detected the expression of MyD88 in order to filter the most effective siRNA plasmid. Constructed recombinant lentivirus by Gateway and determined the titer, then transfected them into endotoxin-stimulated rat alveolar macrophage and the uninfected cells were used as control group, finally determined concentration of IL-1β and IL-6. Results Successfully filtered the most effective siRNA plasmid and constructed recombinant lentivirus. The titer of recombinant lentivirus was 2.0×10^6 TU/ml. IL- 1β and IL-6 of NR8383 cell were significantly reduced after infecting recombinant lentivirus compared with control group. The difference has statistical significance. Conclusion Lentivirus-mediated RNAi can effectively suppress MyD88 expression and significantly reduce the release of cytokine in rat alveolar macrophage, which may provide a possibility that treat rat lung transplantation-related obliterative bronchitis （OB） by targeting antigen-presenting cells（APC） in vivo.