中南大学学报（医学版）
中南大學學報（醫學版）
중남대학학보（의학판）
JOURNAL OF CENTRAL SOUTH UNIVERSITY (MEDICAL SCIENCES)
2009年
10期
970-977
,共8页
宋达疆%黄晓元%杨兴华%肖目张%王双
宋達疆%黃曉元%楊興華%肖目張%王雙
송체강%황효원%양흥화%초목장%왕쌍
RNAi%髓系触发受体-1%慢病毒载体%脆弱类杆菌
RNAi%髓繫觸髮受體-1%慢病毒載體%脆弱類桿菌
RNAi%수계촉발수체-1%만병독재체%취약류간균
RNA interference%triggering receptors expressed on myeloid cells-1%lentiviral vector%Bacteroides fragilis
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DNA oligo, 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 mRNA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.
目的:構建髓繫觸髮受體-1(TREM-1)基因RNAi慢病毒載體,探討TREM-1在脆弱類桿菌所緻炎癥反應中的作用.方法:根據小鼠TREM-1 mRNA序列選擇4箇靶序列併設計閤成4對雙鏈DNA oligo, 其兩耑含酶切位點黏耑,同時閤成1對陰性對照雙鏈DNA oligo;將以上5對雙鏈DNA oligo退火後連入pGCSIL-GFP載體,經PCR及測序鑒定後,將以上質粒分彆與包裝質粒混閤物共轉染293T細胞,包裝產生病毒顆粒.各組病毒載體轉染Raw264.7細胞後,運用real-time PCR和ELISA檢測TREM-1 mRNA和蛋白錶達水平.實時定量PCR和ELISA觀察榦擾TREM-1後脆弱桿菌內毒素引髮的Raw264.7細胞內毒素血癥模型中TREM-1的錶達變化.結果:PCR和測序證實目的雙鏈DNA oligo片段已被準確剋隆到pGCSIL-GFP載體;各質粒與包裝質粒共轉染293T細胞後產生瞭高滴度的慢病毒顆粒;各組慢病毒感染Raw264.7細胞後均可以顯著抑製TREM-1的錶達,以pGCSIL-GFP/Lenti-1組效果最明顯.在脆弱桿菌內毒素引髮的體外細胞內毒素血癥模型中TREM-1錶達明顯增高,實時定量PCR錶明慢病毒榦擾載體能顯著抑製模型中TREM-1的錶達. 結論:成功構建瞭小鼠TREM-1基因RNAi慢病毒載體,所構建的慢病毒載體能夠抑製脆弱類桿菌內毒素所誘導的TREM-1的錶達.
목적:구건수계촉발수체-1(TREM-1)기인RNAi만병독재체,탐토TREM-1재취약류간균소치염증반응중적작용.방법:근거소서TREM-1 mRNA서렬선택4개파서렬병설계합성4대쌍련DNA oligo, 기량단함매절위점점단,동시합성1대음성대조쌍련DNA oligo;장이상5대쌍련DNA oligo퇴화후련입pGCSIL-GFP재체,경PCR급측서감정후,장이상질립분별여포장질립혼합물공전염293T세포,포장산생병독과립.각조병독재체전염Raw264.7세포후,운용real-time PCR화ELISA검측TREM-1 mRNA화단백표체수평.실시정량PCR화ELISA관찰간우TREM-1후취약간균내독소인발적Raw264.7세포내독소혈증모형중TREM-1적표체변화.결과:PCR화측서증실목적쌍련DNA oligo편단이피준학극륭도pGCSIL-GFP재체;각질립여포장질립공전염293T세포후산생료고적도적만병독과립;각조만병독감염Raw264.7세포후균가이현저억제TREM-1적표체,이pGCSIL-GFP/Lenti-1조효과최명현.재취약간균내독소인발적체외세포내독소혈증모형중TREM-1표체명현증고,실시정량PCR표명만병독간우재체능현저억제모형중TREM-1적표체. 결론:성공구건료소서TREM-1기인RNAi만병독재체,소구건적만병독재체능구억제취약류간균내독소소유도적TREM-1적표체.
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.
