摘要
目的:建立NLRP3炎性体缺损的小鼠巨噬细胞模型。方法:构建靶向NLRP3基因的带GFP和Neo标记的shRNA表达载体,转染小鼠巨噬细胞RAW264.7后用G418抗性标记筛选稳定转染的细胞克隆,并利用GFP标记通过流式细胞仪进一步纯化,得到的细胞命名为RAWNKD。利用定量PCR检测RAWNKD细胞及其在LPS和ATP联合刺激下NLRP3基因的稳定抑制效果。结果:RAWNKD细胞GFP标记的阳性率在80%以上,NLRP3基因的抑制率超过90%,即使用LPS和ATP联合刺激NLRP3基因mRNA增加也不明显。结论:成功建立了NLRP3基因稳定敲低的小鼠巨噬细胞系,这种NLRP3炎性体缺损的巨噬细胞是探究NLRP3炎性体活化途径及其介导的炎症信号转导的有力工具。
Objective:To establish a mouse macrophage line lacking NLRP3. Methods:A GFP and neomycin dual selection marker vector which contains an efficient shRNA-coding insert for mouse NLRP3,was constructed and transfected into macrophages(RAW264. 7) to select the stable clone cells in G418-contained medium. Then,the expanded clone cells that retain strong GFP expression were further sorted using the popular flow cytometry. The obtained cell mix( herein termed RAWNKD) were passaged and maintained for further identification,including observation of GFP marker,especially quantitative PCR( RT-q PCR) to confirm knockdown of NLRP3 in the generated RAWNKD cells which were challenged with LPS and ATP or not. Results:Over 80% of RAWNKD cells expressed GFP,and little NLRP3 mRNA was detected in RAWNKD cells,notably no obvious increase in NLRP3 mRNA was observed when the RAWNKD cells were challenged by LPS and ATP. Conclusion:The macrophage line lacking NLRP3 was successfully established,and such macrophage deficient in NLRP3 inflammasome is a valuable cell model for investigating the activation of NLRP3 inflammasome,especially signaling in inflammation mediated by NLRP3 inflammasome.
引文
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