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).Conclusions ADK siRNA modified corneal endothelial cells can induce Treg cells to proliferate and secrete IL-10 and TGF-β,which provides a new method for induction of corneal allograft immune tolerance.%目的 探讨腺苷激酶(ADK)小干扰RNA(siRNA)修饰的角膜内皮细胞诱导调节性T细胞(Treg)增生和分泌的作用.方法 取第3～5代人角膜内皮细胞,实验分为转染组和未转染组,转染组采用Lipofectamine(R) 3000将FAM-ADK siRNA转染进入角膜内皮细胞,采用高效液相色谱法检测各组中腺苷的质量浓度.实验分为对照组[转染对照siRNA]、雷帕霉素组(转染对照siRNA后培养液中加入10 ng/ml雷帕霉素)和ADKsiRNA组(转染ADKsiRNA后培养液中加入10 ng/ml雷帕霉素),采用TUNEL染色检测细胞凋亡比例;采用流式细胞术检测内皮细胞表面细胞间黏附分子-1(ICAM-1)、血管细胞黏附分子-1(VCAM-1)和E-选择素的表达.实验分为单独培养组(单纯淋巴细胞培养)、共培养组(淋巴细胞与角膜内皮细胞共培养)和ADKsiRNA组(淋巴细胞与ADK siRNA转染的角膜内皮细胞共培养),流式细胞术检测各组CD4+ CD25+Foxp3+ Treg细胞的比例;ELISA法检测各组上清液中白细胞介素-10(IL-10)和转化生长因子-β(TGF-β)的质量浓度.结果 流式细胞术检测结果显示,95.1％的角膜内皮细胞表达siRNA,转染组上清液中腺苷的质量浓度为(38.020±6.658) ng/ml,较对照组的(1.663±0.581) ng/ml明显升高,差异有统计学意义(t=5.437,P=0.006).TUNEL染色结果显示,雷帕霉素组角膜内皮细胞平均凋亡率较对照组明显升高,ADK siRNA组角膜内皮细胞平均凋亡率较雷帕霉素组明显降低,差异均有统计学意义(t=3.763,P=0.020;t=4.405,P=0.012).流式细胞术检测结果显示,ADK siRNA组ICAM-1、VCAM-1和E-选择素的平均荧光强度分别为4.060±1.179、3.600±1.234和5.223±1.734,明显弱于对照组的11.600±2.427、11.030 ±2.291和24.270±4.332,差异均有统计学意义(t=2.794,P=0.049;t =2.857,P=0.046;t=4.081,P=0.015).单独培养组、共培养组和ADKsiRNA组淋巴细胞中Treg细胞比例总体比较,差异有统计学意义(F=12.890,P=0.007),其中ADKsiRNA组淋巴细胞中Treg细胞的比例较共培养组明显升高,差异有统计学意义(t=3.650,P=0.022).ELISA检测结果显示,单独培养组、共培养组和ADKsiRNA组淋巴细胞上清液中IL-10和TGF-β的质量浓度总体比较,差异均有统计学意义(F=20.960,P=0.003;F=27.320,P=0.001),其中ADKsiRNA组淋巴细胞上清液中IL-10和TGF-β的质量浓度均较共培养组明显升高,差异均有统计学意义(t=4.492,P=0.011;t=5.280,P=0.006). 结论 ADK siRNA修饰的角膜内皮细胞能够诱导Treg细胞增生及分泌IL-10和TGF-β,为角膜移植的免疫耐受诱导提供了新的选择.
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