IL-35表达对小鼠脾脏CD4^(+)和CD8^(+)T淋巴细胞亚群分化的影响  

Effect of interleukin 35 expression on differentiation of splenic CD4^(+) and CD8^(+)T cell subsets in mice

作　　者：张志强 杨鲁宁 刘恒 王朝霞 张晓宁 ZHANG Zhiqiang;YANG Luning;LIU Heng;WANG Zhaoxia;ZHANG Xiaoning(Department of Pathology,Tianjin Municipal Nankai Hospital,Tianjin 300100,China;Tai’an Detachment Health Team,Shandong Corps of Chinese People’s Armed Police Force,Tai’an,Shandong 271000,China;School of Clinical and Basic Medicine/Institute of Basic Medicine,Shandong First Medical University(Shandong Provincial Academy of Medical Science),Jinan,Shandong 250000,China)

机构地区：[1]天津市南开医院病理科,300100 [2]中国人民武装警察部队山东总队泰安支队卫生队,山东泰安271000 [3]山东第一医科大学(山东省医学科学院)临床与基础医学院(基础医学研究所),济南250000

出　　处：《重庆医学》2023年第14期2115-2120,共6页Chongqing medicine

基　　金：山东省医药卫生科技发展计划项目(202002050100);山东省中医药科技项目(2020M063);山东第一医科大学(山东省医学科学院)青年科学基金培育资助计划项目(202201-016)。

摘　　要：目的构建小鼠白细胞介素(IL)-35基因表达质粒,探讨IL-35表达对小鼠脾脏中CD4^(+)和CD8^(+)T淋巴细胞亚群分化的影响。方法采用PCR法扩增获得小鼠IL-35基因全长片段,将该片段连入鼠干细胞病毒载体(pMSCV-GFP)多克隆位点区,获得携带IL-35基因的质粒载体pMSCV-IL-35-GFP。将9只雄性C57BL/6J小鼠分为3组:PBS组、pMSCV组和pMSCV-IL-35组,分别向3组小鼠尾静脉注射PBS、pMSCV-GFP空载质粒和pMSCV-IL-35-GFP质粒。72 h后分离小鼠脾脏,采用实时荧光定量逆转录PCR(RT-qPCR)检测IL-35亚基EB病毒诱导基因3(Ebi3)和IL-12a mRNA相对表达水平;采用流式细胞术检测CD4^(+)和CD8^(+)T淋巴细胞亚群百分比,并进一步检测CD8^(+)T淋巴细胞免疫功能分子颗粒酶B(Gzmb)和调节性T淋巴细胞(Tregs)细胞特异转录因子叉头/翅膀状螺旋转录因子3(Foxp3)mRNA相对表达水平。结果RT-qPCR结果显示,与PBS组或pMSCV组比较,pMSCV-IL-35组小鼠脾脏Ebi3、IL-12a mRNA相对表达水平均明显升高(P<0.001);流式细胞检测结果显示,与PBS组或pMSCV组比较,pMSCV-IL-35组小鼠脾脏CD8^(+)T淋巴细胞百分比明显降低(P<0.001),而CD4^(+)CD25^(+)Tregs百分比明显升高(P<0.001),并且Gzmb mRNA相对表达水平明显降低(P<0.01),Foxp3 mRNA相对表达水平明显升高(P<0.01)。结论IL-35可以抑制效应性T淋巴细胞的活性,促进Tregs亚群分化发挥免疫调节作用,这将为IL-35基因应用于体内免疫治疗提供理论依据。Objective To construct the mouse interleukin(IL)-35 gene expression plasmid,and to investigate the effects of IL-35 expression on the differentiation of CD4^(+)and CD8^(+)T lymphocytes in mouse spleen.Methods The PCR method was adopted to amplify for obtaining the full-length fragment of mouse IL-35 gene,which was connected into the polyclonal loci region of mouse stem cell viral vector(pMSCV-GFP),and the plasmid vector pMSCV-IL-35-GFP carrying IL-35 gene was obtained.Nine male C57BL/6J mice were divided into three groups:the PBS group,pMSCV group and pMSCV-IL-35 group.The PBS,pMSCV-GFP no-load plasmid and pMSCV-IL-35-GFP plasmid were injected by the tail vein in the three groups,respectively.After 72 h,the mouse spleen was isolated.The real-time reverse transcription-PCR(RT-qPCR)was adopted to detect the relative levels of IL-35 subunit Ebi3 and IL-12a mRNA.The flow cytometer was adopted to detect the percentage of CD4^(+)and CD8^(+)T lymphocytes subsets,and the relative expression levels of CD8^(+)T lymphocyte immune function molecule granzyme B(Gzmb)and regulatory T cells(Tregs)specific transcription factor forkhead/winged helix family transcription factor 3(Foxp3)mRNA were detected.Results The RT-qPCR results showed that compared with the PBS group or pMSCV group,the relative expression levels of mouse spleen Ebi3m and IL-12a mRNA in the pMSCV-IL-35 group were significantly increased(P<0.001).The flow cytometer detection results showed that compared with the PBS group or pMSCV group,the percentage of CD8^(+)T lymphocytes in the pMSCV-IL-35 group was significantly decreased(P<0.001),the percentage of CD4^(+)CD25^(+)Tregs was significantly increased(P<0.001),moreover,the Gzmb mRNA relative expression level was significantly decreased(P<0.01),and the relative expression level of Foxp3 mRNA was significantly increased(P<0.01).Conclusion IL-35 could inhibit the activity of effector T lymphocytes,promote the differentiation of Tregs subset and play the immune regulatory effect,which could provide the theoretical ba

关 键 词：白细胞介素35 调节性T细胞 细胞分化 免疫调节 

分 类 号：R392[医药卫生—免疫学]