摘要
目的探讨PGI2类似物伊洛前列素(Iloprost)对Th17细胞分化的调节作用及其信号机制。方法采用磁珠分选人外周血初始CD4+T细胞,体外诱导其向Th17细胞分化,使用流式细胞术、RT-PCR、ELISA方法分别从Th17细胞频率、RORC m RNA表达以及IL-17A水平3个方面探讨Iloprost对Th17细胞分化的调节作用及受体机制。之后使用免疫荧光法测定胞内c AMP含量,流式细胞术检测胞内STAT3磷酸化水平,以探索Iloprost调节Th17细胞分化的信号通路。结果 Iloprost剂量依赖性地增加了Th17细胞频率和RORC m RNA表达以及IL-17A分泌(P<0.05),而IP受体被阻断之后,Iloprost的上述作用大幅降低。Iloprost可上调Th17细胞胞内c AMP水平,其对Th17细胞分化的调节作用能被c AMP激动剂db-c AMP模拟(P>0.05),且被PKA抑制剂H-89所逆转。另外,Iloprost可上调IL-6诱导的STAT3磷酸化(P<0.05)。同时,db-c AMP模拟了Iloprost对p STAT3的调节作用(P>0.05),而该作用均被H-89抑制。结论 Iloprost通过与IP受体结合,活化c AMP-PKA信号通路,从而上调p STAT3水平,进而促进CD4+T细胞向Th17分化。
The study aims to dissect the role of Iloprost in Th17 cell differentiation and its mechanism. Na?ve CD4+T cells from human peripheral blood were isolated by MACS selection, and cultured under Th17-polarizing condition. We analyzed the effects of Iloprost on Th17 cells differentiation through detecting the proportion of Th17 cells by flow cytometry, the level of RORC m RNA by RT-PCR and the production of IL-17 A by ELISA. In order to explore the mechanism, then, c AMP accumulation assays and flow cytometry were used to detect the level of intracellular c AMP and phosphorylation STAT3. Our results showed that, via IP binding, Iloprost increased the percentage of Th17 cells, RORC m RNA and IL-17 A production. We observed that the regulatory effect of Iloprost were correlated with elevated intracellular c AMP levels, and the effects were mimicked by a c AMP agonist(c AMP), and attenuated by a protein kinase A inhibitor(H-89). On the other hand, our study demonstrated that Iloprost enhanced the activation of STAT3 in response to IL-6. Meanwhile, db-c AMP imitated the above effects of Iloprost, which were weakened by H-89. These results demonstrated that the PGI2-IP interaction can promote the phosphorylation of STAT3, likely via the upregulation of c AMP-PKA signalling, thus facilitates Th17 differentiation.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2016年第1期1-6,共6页
Immunological Journal