RAW264.7细胞M1/M2亚型的诱导和鉴定

Introduction and identification of M1/M2 phenotype of RAW264.7 cells

目的诱导小鼠巨噬细胞RAW264.7分化为M1/M2亚型,并分别根据其标志物的表达情况进行鉴定。方法干预前一天以105个/ml密度铺好细胞,用不同浓度的IFN-γ+LPS干预,刺激分化为M1亚型;用不同浓度IL-4干预,刺激分化为M2亚型。分别于干预后12h提取细胞的总RNA。用realtime PCR方法对M1/M2亚型的标志物iNOS,CD86/MR(CD206),I型精氨酸酶(ArginaseI,Arg-I)进行mRNA水平表达量的鉴定,最终选择最合适的刺激浓度进行后续试验。结果 (1).不同浓度干预试剂刺激12h后,大剂量干预组RAW264.7的细胞状态差,死亡细胞数多,中小剂量组细胞状态良好,完全贴壁,但细胞的形态发生改变;(2).2.5ng/ml IFN-γ+200ng/ml LPS共同作用12h后,RAW264.7的iNOS和CD86表达量最高,较基础水平有明显差异;(3).10ng/ml IL-4作用12h后,RAW264.7的MR(CD206)和ArgI的表达量最高,较基础水平有明显差异。结论用适当浓度的IFN-γ+LPS/IL-4刺激RAW264.7细胞12h,可使其分化为M1/M2亚型。

Objective To induce RAW264.7 cell line to M1/M2 phenotypes and identificate the cytokines expressed on the surface. Methods Plating the cells in 12 wells plate as 105 / ml 24h before intervention and use IFN-γ+LPS to induce M1 phenotype,IL-4 to induce M2 phenotype, with different concertration. Extracting total RNA of cells after intervention. Detecting the surface marker iNOS, CD86/MR,(CD206),ArginaseI (ArgI) of M1/M2 phenotypes, respectively, by realtime PCR on RNA level to select the proper inducing conc...