寡核苷酸骨架对CpG基元诱导人巨噬细胞释放细胞因子的影响

Influence of different oligodeoxynucleotides backbones on cytokines release from human macrophages induced by CpG motifs

目的 探讨不同寡核苷酸骨架对GG 寡核苷酸CpGODNs导致小鼠死亡、诱导细胞因子释放能力的影响。方法 实验前 1h经腹腔注射 6 0 0mg/kgD 氨基半乳糖敏化小鼠 ,采用磷酸硫化CpG 寡核苷酸 (PsCpGODN)、Psnon CpGODN、磷酸二酯CpG 寡核苷酸 (PoCpGODN )和Ponon CpGODN腹腔注射小鼠 ,注射剂量为每只小鼠 10nmol ,观察 7d内小鼠死亡率的差异。体外培养THP 1细胞系 ,在THP 1细胞培养体系中加入上述制剂后 ,37℃、5 %CO2 孵箱中培养 2 0h后离心取上清 ,测定上清中TNF α、IL 6、IL 12的浓度。同时观察CpGODN刺激THP 1细胞 4h后TLR9的表达情况。结果 PsCpGODN可诱导小鼠的死亡以及细胞因子的大量释放 ,而其它三种制剂则无此能力 ;而且PsCpGODN诱导TNF α释放的能力随浓度的增加而增加 ;CpGODN导致的细胞TLR9的表达增加与细胞因子的释放密切相关。结论 磷酸硫代骨架的CpGODN具有导致动物死亡和诱导巨噬细胞大量释放细胞因子的作用 ,磷酸二酯骨架的CpGODN则无此能力。

Objective To investigate the effects of different oligonucleotide backbones on mice lethality and cytokines release induced by CpG ODNs. Methods Mice were sensitized for 1 hour with D-GalN (600 mg/kg) by i.p. before experiment. After Ps CpG ODN, Ps non-CpG ODN, Po CpG ODN and Po non-CpG ODN were injected by i.p. into mice, with the doseage at 10nmol/mouse, Mortality within seven days was observed. THP-1 cell lines were cultivated in vitro. After different agents were added into the culture, cells were cultivated in 5% CO 2 incubator at 37℃ for 20 hours. The supernant was tested for TNF-α, IL-6 and IL-12 level. TLR9 expressions induced by CpG ODN for four hours were measured after THP-1 cell lines were stimulated. Results Ps CpG ODN could lead mice to die and strongly induce the release of cytokines. Others had no such abilities. And the ability to induce the release of TNF-α increased when the concentration increased. TLR9 expression increased, which was associated tightly with cytokines release. Conclusion CpG ODN with phosphorothioate backbone had the ability to induce macrophage to release cytokines strongly and led mice to die, but CpG ODN with phosphodiester backbone had not such ability.