干扰素-α介导系统性红斑狼疮外周血CD56dimCD57+自然杀伤细胞功能的损伤  

Interferon-αmediating the functional damage of CD56 dim CD57+natural killer cells in peripheral blood of systemic lupus erythematosuss

作　　者：赵祥格 刘佳庆 黄会娜 陆智敏 白自然 李霞 祁荆荆 ZHAO Xiang-ge;LIU Jia-qing;HUANG Hui-na;LU Zhi-min;BAI Zi-ran;LI Xia;QI Jing-jing(Department of Immunology,College of Basic Medical Science,Dalian Medical University,Dalian 116044,Liaoning,China)

机构地区：[1]大连医科大学基础医学院免疫学教研室,辽宁大连116044

出　　处：《北京大学学报（医学版）》2023年第6期975-981,共7页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金(82201990、82071834、82271839);大连市青年科技之星项目(2022RQ070)。

摘　　要：目的:探究干扰素-α(interferon-α,IFN-α)对系统性红斑狼疮(systemic lupus erythematosus,SLE)患者外周血CD56^(dim)CD57^(+)自然杀伤(natural killer,NK)细胞凋亡和杀伤功能的调控作用及具体机制。方法:选取大连医科大学附属第二医院就诊的64例初治SLE患者和16例健康人(healthy control,HC)为研究对象,采用实时荧光定量聚合酶链式反应检测NK细胞杀伤功能相关分子的基因表达水平;将CD56^(dim)CD57^(+)NK细胞与K562细胞共培养后,采用膜联蛋白和7氨基放线菌素标记凋亡的K562细胞;用浓度分别为20、40、80μmol/L的过氧化氢(hydrogen peroxide,H2O2)处理外周血单个核细胞,并以不使用H2O2处理作为对照组,采用流式细胞术检测穿孔素(per-forin,PRF)表达水平;采用酶联免疫吸附试验测定血清中IFN-α的浓度;采用流式细胞术检测CD56^(dim)CD57^(+)NK细胞表面IFN-α受体(interferon-αreceptor,IFNAR)表达水平,并用平均荧光强度(mean fluorescence intensity,MFI)表示;用浓度为1000 U/mL的IFN-α处理CD56^(dim)CD57^(+)NK细胞24、48、72 h,并以不使用IFN-α处理作为对照组,采用流式细胞术检测细胞凋亡及线粒体活性氧(mitochondrial reactive oxygen species,mtROS)表达水平,并用MFI表示。结果:与HC(n=3)相比,SLE患者(n=3)外周血总NK细胞的PRF1基因表达水平降低(1.24±0.41 vs.0.57±0.12,P=0.05)。与HC(n=5)相比,SLE患者(n=5)外周血CD56^(dim)CD57^(+)NK细胞杀伤K562细胞的能力明显下降(58.61%±10.60%vs.36.74%±6.27%,P<0.01)。与对照组(n=5,97.51%±1.67%)相比,不同浓度的H2O2处理可显著下调CD56^(dim)CD57^(+)NK细胞的PRF表达水平且呈剂量依赖性,浓度为20μmol/L的H2O2组PRF为83.23%±8.48%(n=5,P<0.05),浓度为40μmol/L的H2O2组PRF为79.53%±8.56%(n=5,P<0.01),浓度为80μmol/L的H2O2组PRF为76.67%±7.16%(n=5,P<0.01)。与HC(n=16)相比,系统性红斑狼疮疾病活动评分(systemic lupus erythematosus disease activity index,SLEDAI)为中高疾病活动度(SLEDAI≥10)的SLEObjective:To investigate the regulatory effect of interferon-α(IFN-α)on the apoptosis and killing function of CD56^(dim)CD57^(+)natural killer(NK)cells in systemic lupus erythematosus(SLE)patients,and to explore the specific mechanism.Methods:A total of sixty-four newly treated SLE patients and sixteen healthy controls(HC)enrolled in the Second Hospital of Dalian Medical University were selected as the research subjects.And the gene expression levels of molecules related to NK cell-killing function were detected by real-time quantitative polymerase chain reaction.CD56^(dim)CD57^(+)NK cells were co-cultured with the K562 cells,and the apoptotic K562 cells were labeled with Annexin-Ⅴand 7-amino-actinomycin D.Peripheral blood mononuclear cells were treated with 20,40,and 80μmol/L hydrogen peroxide(H 2O 2),and treated without H 2O 2 as control,the expression level of perforin(PRF)was detected by flow cytometry.The concentration of IFN-αin serum was determined by enzyme linked immunosorbent assay.The expression levels of IFN-αreceptors(IFNAR)on the surface of CD56^(dim)CD57^(+)NK cells were detected by flow cytometry,and were represented by mean fluorescence intensity(MFI).CD56^(dim)CD57^(+)NK cells were treated with 1000 U/mL IFN-αfor 24,48 and 72 h,and no IFN-αtreatment was used as the control,the apoptosis and the expression levels of mitochondrial reactive oxygen species(mtROS)were measured by flow cytometry and represented by MFI.Results:Compared with HC(n=3),the expression levels of PRF1 gene in peripheral blood NK cells of the SLE patients(n=3)were decreased(1.24±0.41 vs.0.57±0.12,P=0.05).Compared with HC(n=5),the ability of peripheral blood CD56^(dim)CD57^(+)NK cells in the SLE patients(n=5)to kill K562 cells was significantly decreased(58.61%±10.60%vs.36.74%±6.27%,P<0.01).Compared with the control(n=5,97.51%±1.67%),different concentrations of H 2O 2 treatment significantly down-regulated the PRF expression levels of CD56^(dim)CD57^(+)NK cells in a dose-dependent manner,the 20μmol/L H 2O 2 PRF w

关 键 词：系统性红斑狼疮 自然杀伤细胞 干扰素-Α 活性氧 穿孔素 

分 类 号：R593.2[医药卫生—内科学]