RAW264-7细胞M1-M2亚型的诱导和鉴定.pdf

Research Articles 117 RAW264.7细胞M1/M2亚型的诱导和鉴定 5 【摘要】 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 10 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。 【关键词】RAW264.7 细胞；M1/M2 亚型；炎症；动脉粥样硬化 Introduction and Identifi cation of M1/M2 Phenotype of RAW264.7 Cells CHEN Tao1,LIANG Xiao1,YUAN Zuyi1,1Department of Cardiovascular Medicine,First Affi liated Hospital,Key Laboratory of Environment and Gene Related Disease of Ministry Education,Xi’an Jiaotong University College of Medicine,Xi’an,710061,China. 【Abstract】Objective To induce RAW264.7 cell line to M1/M2 phenotypes and identifi cate 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 concertration. Results (1). Cells retain well growth condition with inventional reagents of middle and smallest concertation but highest concertation; (2)