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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。
Objective To investigate the regulatory effect of PGI2 analog Iloprost on Th17 cell differentiation and its signal mechanism. Methods The primary CD4 + T cells were sorted by magnetic beads and induced into Th17 cells in vitro. The expression of Th17 cells, the expression of RORC mRNA and the level of IL-17A were detected by flow cytometry, RT-PCR and ELISA. To investigate the regulatory effect of Iloprost on Th17 cell differentiation and its receptor mechanism. After intracellular cAMP levels were measured by immunofluorescence, intracellular phosphorylation of STAT3 was detected by flow cytometry to explore the signaling pathway by which Iloprost regulates Th17 cell differentiation. Results Iloprost increased the frequency of Th17 cells and the expression of RORC m RNA and the secretion of IL-17A (P <0.05) in a dose-dependent manner. However, the effect of Iloprost was significantly reduced after IP receptor was blocked. Iloprost up-regulated the intracellular c AMP level in Th17 cells. The effect of Iloprost on Th17 cell differentiation was mimicked by cAMP agonist db-c AMP (P> 0.05) and reversed by PKA inhibitor H-89. In addition, Iloprost up-regulated IL-6-induced STAT3 phosphorylation (P <0.05). At the same time, db-c AMP mimicked the regulation of p-STAT3 by Iloprost (P> 0.05), and this effect was inhibited by H-89. Conclusion Iloprost can activate c AMP-PKA signal pathway by binding to IP receptor, thereby up-regulating the level of p-STAT3, thereby promoting the differentiation of CD4 + T cells to Th17 cells.