组蛋白的制备及其对巨噬细胞的影响

Preparation of histones and their effects on macrophages

目的:用不同方法制备组蛋白并观察组蛋白在体外对巨噬细胞活力及细胞因子释放的影响。方法:用基因克隆的方法构建组蛋白H3和H4的原核表达质粒,分别表达和纯化带谷胱甘肽S-转移酶(GST)和组氨酸(His)标签的组蛋白(GST-H3、GST-H4、His-H3和His-H4);用高盐法提取真核细胞(RAW264.7和293F细胞)组蛋白;用上述不同来源组蛋白(7.5～50 mg/L)刺激巨噬细胞4 h,利用MTT和流式细胞术检测巨噬细胞活力;用液相芯片方法检测不同来源组蛋白对巨噬细胞释放在培养上清中的细胞因子白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)的浓度。结果:His-H3/H4和真核细胞来源的组蛋白明显降低细胞活性,诱导RAW264.7细胞发生晚期凋亡和坏死;不同来源组蛋白对RAW264.7细胞释放IL-6和TNF-α均有不同程度的促进作用。结论:原核表达His-H3、His-H4及真核细胞提取的组蛋白均可抑制巨噬细胞活力,诱导巨噬细胞发生晚期凋亡和坏死。组蛋白可能通过促进炎症细胞因子的释放参与了炎症反应过程。

AIM： To prepare histones with different methods and to observe their effects on cell viability and cytokine release by macrophages in vitro. METHODS ： Prokaryotic expression plasmids of histone H3 and histone H4 were constructed by cloning methods. The histones containing GST-tag or His-tag （GST-H3, GST-H3, His-H3, His-H4） were expressed and purified. The histones from eukaryotic ceils （ RAW264.7 and 293F cells） were extracted with the high salt method. RAW264. 7 cells were treated with histones （7. 5 -50 mg/L） for 4 h and the cell vitality was examined by MTY assay and flow cytometry. The concentrations of IL-6 and TNF-ot in the culture supernatants of RAW264. 7 cells treated with histones were also detected. RESULTS： His-H3/H4 and the histones from eukaryotic cells significantly reduced the viability of RAW264. 7 cells and induced apoptosis. The effects of histones from different sources on the releases of IL-6 and TNF-a were different. CONCLUSION： His-H3/His-H4 expressed by prokaryotic plasmids and the histones extracted from eukaryotic cells affect the vitality of macrophages as well as induce late apoptosis and necrosis. Histone may involve in the inflammatory process by promoting the release of inflammatory cytokines.