CAJ | 학술논문

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。
목적：구건 Toll양수체(TLR)4기인만병독표체재체개도적소간우 RNA(siRNA),관찰기대대서폐포거서세포NR8383 TLR4적침묵효응。방법설계4조침대TLR4기인적siRNA파서렬,경합성、퇴화、매절,여선성재체GV115련접,전입세균감수태세포,경취합매련반응(PCR)사선양성극륭、측서감정。공전염293T세포,포장산생만병독과립,분별감염NR8383세포,실시정량PCR(RT-PCR)검측NR8383세포TLR4 mRNA적표체。근거사선결과,선취최유효적재체진행병독대량포장。결과경측서험증,만병독재체구건성공병획득상응적만병독,NR8383세포감염만병독후,TLR4기인mRNA표체명현하강,LV-TLR4-RNAi(Tgt-2)작용교명현,고감효솔체도65%。결론성공구건침대파향대서TLR4기인적RNAi만병독재체,위진일보연구TLR4신호통로재급성폐손상중적작용전정기출。
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 siRNA 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 mRNA 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.