白细胞介素-35对肝细胞癌患者CD4^(+)CD25^(+)CD127^(dim/-)调节性T细胞的调控作用  

Regulation of Interleukin-35 to CD4^(+)CD25^(+)CD127^(dim/-) Regulatory T Cells in Patients with Hepatocellular Carcinoma

作　　者：李彧[1] 李连香[1] 奚逢瑜[1] 薛敬东 叶苗青 杨跃青 党珊[1] 张鸿[1] 段降龙[1] 高瑛[1] LI Yu;LI Lianxiang;XI Fengyu(Shaanxi People's Hospital,Xi'an,710068)

机构地区：[1]陕西省人民医院,710068 [2]陕西省中医医院,710003

出　　处：《实用癌症杂志》2023年第8期1239-1244,共6页The Practical Journal of Cancer

基　　金：陕西省重点研发计划(编号:2021SF-236);陕西省科技创新团队(编号:2021TD-40)。

摘　　要：目的 观察肝细胞肝癌(HCC)患者外周血白细胞介素-35(IL-35)水平和CD4^(+)CD25^(+)CD127^(dim/-)调节性T细胞(Treg)比例,评估IL-35对Treg数量和功能的影响。方法 共有51例HCC患者和25例对照者,分离外周血单个核细胞(PBMC)和血浆,实时定量PCR法检测PBMC中IL-35亚基mRNA相对表达量,酶联免疫吸附试验(ELISA)法检测外周血IL-35水平,流式细胞术检测PBMC中CD4^(+)CD25^(+)CD127^(dim/-)Treg比例。使用重组人IL-35(1 ng/ml)刺激HCC组PBMC 24 h,观察Treg比例和EBI3、IL-12p35 mRNA相对表达量的变化。分选17例HCC患者的Treg,经重组人IL-35刺激后与自体PBMC共培养48 h,通过检测细胞增殖和细胞因子表达评估IL-35对Treg功能的影响。组间比较采用t检验或配对t检验。结果 HCC组血浆IL-35水平显著高于对照组[(1024±218.0)pg/ml比(297.6±77.78)pg/ml,t=16.12,P<0.0001)],HCC组PBMC中EBI3和IL-12p35 mRNA相对表达量均显著高于NC组(P<0.0001)。HCC组CD4^(+)CD25^(+)CD127^(dim/-)Treg比例显著高于对照组[(6.35±1.46)%比(4.55±0.90)%,t=5.641,P<0.0001)],HCC组中CD4^(+)CD25^(+)CD127^(dim/-)Treg比例与血浆IL-35水平呈显著正相关(γ=0.400,P=0.0036)。HCC组PBMC经IL-35刺激后CD4^(+)CD25^(+)CD127^(dim/-)Treg比例较无IL-35刺激升高(P=0.041),EBI3和IL-12p35 mRNA相对表达量亦升高(P<0.001)。HCC患者纯化的CD4^(+)CD25^(+)CD127^(dim/-)Treg经IL-35刺激后,免疫检查点受体表达升高(P<0.05),抑制自体PBMC增殖的能力增强,共培养系统中细胞数量减少(P=0.023)。IFN-γ分泌水平在经IL-35刺激的Treg中显著降低(P=0.0002),而IL-10和IL-35分泌水平在经IL-35刺激的Treg中显著升高(P<0.05)。结论 HCC患者中IL-35水平升高,增强Treg免疫抑制活性。Objective To investigate the circulating IL-35 level and CD4^(+)CD25^(+)CD127^(dim/-)regulatory T cells(Treg) percentage in hepatocellular carcinoma(HCC),and to assess the influence of IL-35 to number and function of Treg.Methods 51 HCC patients and 25 controls participated in the study.Plasma and peripheral blood mononuclear cells(PBMC) were isolated.Plasma IL-35 level was measured by enzyme-linked immunosorbent assay(ELISA).EBI3 and IL-12p35 mRNA relative levels were measured by real-time PCR.CD4^(+)CD25^(+)CD127^(dim/-)Treg percentage was measured by flow cytometry.PBMC from HCC patients was stimulated with recombinant human IL-35(1ng/ml) for 24 h.Treg percentage and EBI3 and IL-12p35 mRNA relative levels were investigated.CD4^(+)CD25^(+)CD127^(dim/-)Treg from seventeen HCC patients was purified using magnetic activated cell sorting method,and was stimulated with recombinant human IL-35.Stimulated Treg was co-cultured with autogolous PBMC for 48 h.The influence of IL-35 to Treg function was assessed by measuring cellular proliferation and cytokine production.Student t test or paired t test was used for comparison between two groups.Results Plasma IL-35 level was increased in HCC patients compared with controls(1024±218.0 pg/ml vs 297.6±77.78 pg/ml,t=16.12,P<0.0001).EBI3 and IL-12p35 mRNA relative levels were also increased in HCC patients compared with controls(P<0.0001).CD4^(+)CD25^(+)CD127^(dim/-)Treg percentage was elevated in HCC patients compared with controls(6.35±1.46% vs 4.55±0.90%,t=5.641,P<0.0001).CD4^(+)CD25^(+)CD127^(dim/-)Treg percentage was positively correlated with plasma IL-35 level in HCC patients(γ=0.400,P=0.0036).IL-35 stimulation elevated CD4^(+)CD25^(+)CD127^(dim/-)Treg percentage in PBMC from HCC patients(P=0.041).Meanwhile EBI3 and IL-12p35 mRNA relative levels were also increased in response to IL-35 stimulation(P<0.001).IL-35 stimulation to purified CD4^(+)CD25^(+)CD127^(dim/-)Treg not only elevated immune checkpoint receptors expression(P<0.05),but also promoted inhibitory funct

关 键 词：肝细胞肝癌 白细胞介素-35 调节性T细胞 

分 类 号：R735.7[医药卫生—肿瘤]