中文摘要：

目的：构建髓系触发受体-1（TREM-1）基因RNAi慢病毒载体,探讨TREM-1在脆弱类杆菌所致炎症反应中的作用。方法：根据小鼠TREM-1 mRNA序列选择4个靶序列并设计合成4对双链DNAoligo,其两端含酶切位点黏端,同时合成1对阴性对照双链DNA oligo;将以上5对双链DNA oligo退火后连入pGCSIL-GFP载体,经PCR及测序鉴定后,将以上质粒分别与包装质粒混合物共转染293T细胞,包装产生病毒颗粒。各组病毒载体转染Raw264.7细胞后,运用real-time PCR和ELISA检测TREM-1 mR-NA和蛋白表达水平。实时定量PCR和ELISA观察干扰TREM-1后脆弱杆菌内毒素引发的Raw264.7细胞内毒素血症模型中TREM-1的表达变化。结果：PCR和测序证实目的双链DNA oligo片段已被准确克隆到pGCSIL-GFP载体;各质粒与包装质粒共转染293T细胞后产生了高滴度的慢病毒颗粒;各组慢病毒感染Raw264.7细胞后均可以显著抑制TREM-1的表达,以pGCSIL-GFP/Lenti-1组效果最明显。在脆弱杆菌内毒素引发的体外细胞内毒素血症模型中TREM-1表达明显增高,实时定量PCR表明慢病毒干扰载体能显著抑制模型中TREM-1的表达。结论：成功构建了小鼠TREM-1基因RNAi慢病毒载体,所构建的慢病毒载体能够抑制脆弱类杆菌内毒素所诱导的TREM-1的表达。

英文摘要：

Objective To construct a lentiviral vector of RNA interference（RNAi） of murine triggering receptor expressed on myeloid cells-1（TREM-1） gene and to explore the effect of TREM-1 on the inflammatory response caused by Bacteroides fragilis.Methods Four target sequences were selected according to murine TREM-1 mRNA sequence,and then 4 pairs of double-strand DNA oligo according to these target sequences and one pair of negative control double-strand DNA oligo were designed and synthesized.These fragments were subcloned into pGCSIL-GFP/Lenti plasmid.After being identified by PCR and sequencing,these plasmids were cotransfected into 293T cells to package lentiviral particles.The lentiviral vector particles were transfected into Raw 264.7 cells and TREM-1 expression in the transfected cells was assayed by real-time PCR and ELISA.Different concentrations of Bacteroides fragilis lipopolysaccaride（LPS） were administered in the Raw264.7 cells,and the cells were stimulated with LPS for 12 h.TREM-1 expression was determined by real-time PCR and ELISA at the time points.Results PCR and sequencing confirmed that lentiviral vectors had the correct structure and could express high titer of virus.After being transfected into Raw264.7 cells,TREM-1 expression was knocked down significantly by all of these lentiviral vectors at both protein and mRNA levels,and the pGCSIL-GFP/Lenti-1 had the most efficient interference.TREM-1 was upregulated in the presence of Bacteroides fragilis LPS,and this increase was partly abrogated in the TREM-1 siRNA-treated cell models of endotoxemia,depending on the sequence.Conclusion The lentivirus RNAi vector of TREM-1 was constructed successfully.The lentivirus RNAi vector of TREM-1 can inhibit the expression of TREM-1 in the murine endotoxemia model caused by Bacteroides fragilis LPS.