摘要
目的探讨酸敏感离子通道3(ASIC3)通过调控Ca^(2+)-钙调磷酸酶(CaN)-活化T细胞核因子(NFAT)信号轴对调节性T细胞(Treg)/辅助性T细胞17(Th17)免疫平衡的作用机制。方法将供者血小板与受者外周血单个核细胞(PBMCs)按250∶1比例进行体外共培养模拟体内血小板输注状态,通过构建ASIC3过表达慢病毒,与体外细胞模型共培养,将其分为三组:(1)空白对照组;(2)阴性对照慢病毒组;(3)ASIC3过表达慢病毒组。采用流式细胞术检测Treg(Foxp3+)和Th17(IL-17A+)细胞比例及细胞内Ca^(2+)水平;Western blot检测ASIC3、磷脂酶CB(PLCB)、CaN、NFATC1及其磷酸化蛋白(p-NFATC1)的表达。结果与空白对照组相比,ASIC3过表达慢病毒组Th17细胞比例显著升高(P<0.01),Treg比例显著降低(P<0.05);细胞内Ca^(2+)浓度升高1.8倍(P<0.001)。蛋白检测显示,ASIC3过表达组PLCB、CaN及p-NFATC1表达均显著上调(均P<0.05)。结论ASIC3过表达通过激活Ca^(2+)-CaN-NFAT信号轴,上调PLCB/CaN/p-NFATC1表达,促进NFAT持续性活化,进而驱动IL-17转录,最终导致Treg/Th17免疫失衡。
Objective To investigate the regulatory mechanism underlying the effect of acid-sensing ion channel 3(ASIC3)on the balance between regulatory T(Treg)cells and T helper type 17(Th17)cells via the Ca^(2+)-calcineurin(CaN)-nuclear factor of activated T cells(NFAT)signaling axis.Methods Donor platelets and recipient peripheral blood mononuclear cells(PBMCs)were co-cultured in vitro at a ratio of 250∶1 to simulate the state of platelet transfusion in vitro.By constructing ASIC3 overexpression lentivirus and co-culturing with in vitro cell model,they were divided into three groups:(1)blank control group;(2)negative control lentivirus group;(3)ASIC3 overexpression lentivirus group.The proportion of Treg(Foxp3+)and Th17(IL-17A+)cells and intracellular Ca^(2+)level were detected by flow cytometry.The expression of ASIC3,phospholipase CB(PLCB),CaN,NFATC1 and its phosphorylated protein(p-NFATC1)were detected by Western blot.Results Compared with the blank control group,the proportion of Th17 cells in the ASIC3 overexpression lentivirus group was significantly increased(P<0.01),and the proportion of Treg cells was significantly decreased(P<0.05).Intracellular Ca^(2+)concentration increased 1.8 times(P<0.001).Protein detection showed that the expression of PLCB,CaNand p-NFATC1 in ASIC3 overexpression group were significantly up-regulated(all P<0.05).Conclusion ASIC3 overexpression up-regulates the expression of PLCB/CaN/p-NFATC1 by activating the Ca^(2+)-CaN-NFAT signaling axis,promotes the continuous activation of NFAT,and then drives IL-17 transcription,eventually leading to Treg/Th17 immune imbalance.
作者
梁静
范娜
郑淑贤
刘雯
LIANG Jing;FAN Na;ZHENG Shuxian;LIU Wen(The Sixth Affiliated Hospital of Xinjiang Medical University,Urumqi 830002)
出处
《临床输血与检验》
2025年第6期782-788,共7页
Journal of Clinical Transfusion and Laboratory Medicine
基金
新疆维吾尔自治区自然科学基金面上项目(No.2022D01C585)
新疆维吾尔自治区自然科学基金青年基金项目(No.2022D01C822)资助。
