Effect of adenosine kinase small interfering RNA modified corneal endothelial cells on the proliferation and secretion of regulatory T cells

Objective

To investigate the role of adenosine kinase (ADK)small interfering RNA(siRNA) modified corneal endothelial cells on the proliferation and secretion of regulatory T cells (Treg cells).

Methods

The experiment was divided into transfected group and non-transfected group, FAM-ADK siRNA was transfected into corneal endothelial cells with Lipofectamine 3000 in transfected group, and the average mass concentration of adenosine was detected by high performance liquid chromatography in both groups.The experiment was divided into control group (transfected with control siRNA), rapamycin group (added 10 ng/ml rapamycin to culture supernatant after transfected with control siRNA) and ADK siRNA group (added 10 ng/ml rapamycin to culture supernatant after transfected with ADK siRNA). TUNEL staining was used to detect the proportion of apoptosis.Flow cytometry was used to detect the expressions of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin in corneal endothelial cells.The cells were divided into single cultured group (lymphocytes were cultured separately), co-culture group (lymphocytes were co-cultured with corneal endothelial cells) and ADK siRNA group (lymphocytes were co-cultured with ADK siRNA transfected corneal endothelial cells), and the proportion of CD4⁺ CD25⁺ Foxp3⁺ Treg cells was detected by flow cytometry, and the content of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) in the supernatant was detected by ELISA in the three groups.The access and use of clinical specimens was approved by Ethic Committee of Tianjing Taida Hospital (TD201705140901).

Results

Flow cytometry showed that 95.1% of the endothelial cells expressed siRNA.The average mass concentration of adenosine in the supernatant of the transfection group was significantly higher than that in the control group ([38.020±6.658]ng/ml vs.[1.663±0.581]ng/ml)(t=5.437, P=0.006). TUNEL staining showed that the average apoptotic cell proportion of corneal endothelial cells in the rapamycin group was significantly higher than that in the control group, and the average apoptotic cell proportion of corneal endothelial cells in the ADK siRNA group was significantly lower than that in the rapamycin group, with significant differences between them (t=3.763, P=0.020; t=4.405, P=0.012). Flow cytometry showed that the average fluorescence intensities of ICAM-1, VCAM-1 and E-selectin in the ADK siRNA group was weaker than that in the control group (4.060±1.179 vs.11.600±2.427, 3.600±1.234 vs.11.030±2.291, 5.223±1.734 vs.24.270±4.332), with significant differences between them (t=2.794, P=0.049; t=2.857, P=0.046; t=4.081, P=0.015). The proportions of Treg cells in the single cultured group, co-culture group and ADK siRNA group was significantly different (F=12.890, P=0.007), and the proportion of Treg cells in the ADK siRNA group was significantly lower than that in the co-culture group (t=3.650, P=0.022). ELISA assay showed that, the contents of IL-10 and TGF-β in the supernatant in the single cultured group, co-culture group and ADK siRNA group were significantly different (F=20.960, P=0.003; F=27.320, P=0.001), and the content of IL-10 and TGF-β in the supernatant in the ADK siRNA group was significantly higher than that in the co-culture group, respectively (t=4.492, P=0.011; t=5.280, P=0.006).

Key words: Adenosine kinase; Corneal transplantation; Endothelial cells; Regulatory T cells