维生素D诱导调节性T细胞分化及其在自身免疫性肝炎发病中的作用

摘要

目的 分析自身免疫性肝炎(AIH)患者外周血调节性T细胞(Treg)比例、白细胞介素-10(IL-10)水平与维生素D的相关性,探讨维生素D对Treg细胞分化的影响.方法 选取平顶山市第一人民医院2015年2月至2016年12月收治的AIH患者30例为观察组,另选取同期体检健康者18例为对照组,对照组于体检当日、观察组于入院次日抽取晨起空腹肘静脉血10 mL,以肝素锂抗凝,分离血浆冻存,并采用密度梯度离心法分离外周血单个核细胞(PB-MC),分别采用全自动生物化学分析仪和酶联免疫吸附试验(ELISA)检测血浆25-羟维生素D[25-(OH)-D3]和IL-10水平,采用多色荧光抗体结合流式细胞术分析样本中CD4+ CD25+ Foxp3+ IL-10+ Treg细胞比例,分析三者之间的相关性.在体外条件下分离观察组患者的PBMC,在有或无活化形式的维生素D[1,25-(OH)2-D3]的条件下给予佛波酯(PMA)刺激16 h,再用流式细胞术检测刺激后PBMC中CD4+ CD25+ Foxp3+ IL-10+ Treg细胞比例,同时采用ELISA法检测刺激后上清液中IL-10水平.结果 观察组患者血浆25-(OH)-D3、IL-10水平显著低于对照组(P ＜0.05,P＜0.01).观察组患者外周血CD4+ CD25+ Foxp3+ IL-10+Treg细胞比例显著低于对照组(P＜0.05).观察组患者外周血25-(OH)-D3水平与IL-10水平及CD4+ CD25+ Foxp3+ IL-10+ Treg细胞比例均呈正相关(R2=0.379,P=0.0003;R2=0-151,P=0.034 1).在有1,25-(OH)2-D3的条件下给予PMA刺激,观察组患者的CD4+ CD25+ Foxp3+ IL-10+ Treg细胞比例和上清液中IL-10水平均显著高于无1,25-(OH)2-D3的条件下给予PMA刺激后(P＜0.05).结论 维生素D可直接诱导Treg细胞增殖分化,AIH患者体内Treg细胞减少在某种程度上与维生素D缺乏有关.%Objective To investigate the relationship between the proportion of regulatory T (Treg) cells,interleukin10 (IL-10) level and vitamin D in peripheral blood of patients with autoimmune hepatitis (AIH),and to investigate the effect of vitamin D on the differentiation of Treg cells.Methods Thirty patients with AIH as observation group in the First People's Hospital of Pingdingshan City from February 2015 to December 2016 were selected and eighteen healthy subjects were selected as control group.The ulnar venous blood samples 10 mL of the subjects in the control group and observation group were collected on the day of medical examination or the next day after admission.The blood samples were anticoagulated with heparin lithium,and the plasma was separated and cryopreserved.The peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation.The levels of plasma 25-hydroxy vitamin D [25-(OH)-D3] and IL-10 were detected by automatic biochemical analyzer and enzyme linked immunosorbent assay (ELISA).The proportion of CD4 + CD25 + Foxp3 + IL-10 + Treg cells in the samples was detected by multicolor fluorescent antibody combined with flow cytometry.The correlation among them was analyzed.The PBMC were isolated in vitro in the observation group,then the PBMC were stimulated with phorbol ester (PMA) for 16 hours under the conditions with or without the activated vitamin D.Then the proportion of CD4 + CD25 + Foxp3 + IL-1 0 + Treg cells was detected by flow cytometry,and the level of IL-10 in supernatant was determined by ELISA.Results The levels of plasma 25-(OH)-D3 and IL-10 in the observation group were significantly lower than those in the control group (P ＜ 0.05,P ＜ 0.01).The proportion of peripheral blood CD4 + CD25 + Foxp3 + IL-10 + Treg cells in the observation group was significantly lower than that in the control group (P ＜ 0.05).The peripheral blood 25-(OH)-D3 level was positively correlated with the proportion of IL-10 and CD4 + CD25 + Foxp3 + IL-10 + Treg cells in the observation group (R2 =0.379,P =0.000 3;R2 =0.151,P =0.034 1).After PMA stimulation,the levels of CD4 + CD25 + Foxp3 + IL-10 + Treg cell and IL-10 in supernatant under the condition of 1,25-(OH) 2-D3 were significantly higher than those under the condition of absenting 1,25-(OH) 2-D3 in the observation group (P ＜ 0.05).Conclusion Vitamin D can directly induce the proliferation and differentiation of Treg cells.The decrease of Treg cells in AIH patients is related to vitamin D deficiency in some extent.