关键词:

UCMSCs/PBMCs 共培养系统, 肝脏免疫微环境, RNA?seq, Th17/Treg 平衡, TGF?β 信号通路

Abstract:

Objective To investigate the impact and potential regulatory mechanisms of Umbilical Cord Mesenchymal Stem Cells（UCMSCs）on the immune microenvironment of autoimmune hepatitis（AIH）. Methods Peripheral blood mononuclear cells（PBMCs）from AIH patients co⁃cultured with UCMSCs at a ratio of 10∶1 （Co⁃culture 1）or 5∶1（Co⁃culture 2），respectively. Flow cytometry was used to detect Th17，Treg and Th17/Treg ratio；ELISA was used to detect the levels of transforming growth factor β1（TGF ⁃β1），interleukin ⁃17（IL ⁃17） and IL ⁃ 6 in the supernatant. RNA ⁃ seq was used to screen the differential genes before and after co ⁃ culture of PBMCs，and the top 15 most highly expressed mRNAs were enriched for subsequent bioinformatic analysis. western blot and RT⁃qPCR were used to determine the expression levels of Foxp3 and RORγt. Results UCMSCs significantly decreased CD3+ CD8⁃IL⁃17A+ Th17 ratio and increased CD25+ Foxp3+ CD4+ Treg ratio. Compared with the PBMCs group，TGF⁃β1 was significantly upregulated（P < 0.01）and IL⁃17 and IL⁃6 were significantly downregu⁃ lated（P < 0.05）in the Co ⁃culture 2 group. Transcriptome sequencing analysis revealed that the transcription factors were mainly focused on immune response and T cell activation；among them，SMAD3 and STAT3 occupied a more central position. Protein and mRNA assays showed that the expression levels of Foxp3 and SMAD3 were significantly upregulated in the Co⁃culture 2 group（P < 0.01），and the expression levels of RORγt and STAT3 were significantly downregulated（P < 0.01）. Conclusion UCMSCs promote the differentiation of Th17 to Treg cells through the TGF⁃β pathway，suppress inflammatory responses to improve the hepatic immune microenvironment.

Key words:

UCMSCs/PBMCs co?culture system, liver immune microenvironment, RNA?seq, Th17/ Treg homeostasis, TGF?β signaling pathway