Lenti-Toll样受体4-siRNA的构建及对大鼠肺泡巨噬细胞NR8383的影响

Construction of Lenti-TLR4-siRNA and its effect on rat alveolar macrophage NR8383

目的构建Toll样受体(TLR)4基因慢病毒表达载体介导的小干扰RNA(siRNA),观察其对大鼠肺泡巨噬细胞NR8383 TLR4的沉默效应。方法设计4条针对TLR4基因的siRNA靶序列,经合成、退火、酶切,与线性载体GV115连接,转入细菌感受态细胞,经聚合酶链反应(PCR)筛选阳性克隆、测序鉴定。共转染293T细胞,包装产生慢病毒颗粒,分别感染NR8383细胞,实时定量PCR(RT-PCR)检测NR8383细胞TLR4 mRNA的表达。根据筛选结果,选取最有效的载体进行病毒大量包装。结果经测序验证,慢病毒载体构建成功并获得相应的慢病毒,NR8383细胞感染慢病毒后,TLR4基因mRNA表达明显下降,LV-TLR4-RNAi(Tgt-2)作用较明显,敲减效率达到65%。结论成功构建针对靶向大鼠TLR4基因的RNAi慢病毒载体,为进一步研究TLR4信号通路在急性肺损伤中的作用奠定基础。

Objective To construct the lentiviral vector-mediated small interfering RNA（siRNA） of TLR4, and examine the silent effect on the rat alveolar macrophage NR8383. Methods 4 si RNA sequences targeting TLR4 gene were designed. After synthesis, annealing and restriction enzyme digestion, TLR4-RNAi were ligated with GV115,transformed into competent bacterial cells, and confirmed by PCR and DNA sequencing. 293 T cells were co-transfected.The four kinds of recombinant lentiviruses were used to infect NR8383 cells, and the expression levels of TLR4 m RNA were detected by RT-PCR. According to the result, the most effective vector was selected for viruses amounts of packaging. Results 4 Lenti-TLR4-siRNA sequences were successfully inserted into the lentiviral vectors. The TLR4 expression in NR8383 cells infected with lentiviral vectors was significantly inhibited at mRNA levels when compared with that in the non-transfected and empty vector-transfected NR 8383 cells. The effect was most significant in NR8383 cells infected with LV-TLR4-RNAi（Tgt-2）. The TLR4 mRNA expression was decreased by 65%. Conclusion Successful construction of lentiviral vector RNAi on rat with targeted TLR4 gene lay the foundation for further studying the role of TLR4 signaling pathway in acute lung injury.