摘要
目的探讨脓毒症小鼠CD4＋CD25＋Treg（regulatory cell）细胞中microRNA-10a（miR-10a）表达规律及与细胞免疫功能的关系。方法采用清沽级雄性Balb／c小鼠建立稳定的小鼠盲肠结扎穿孔（ cecal ligation and puncture, CLP ）模型，分别于术后1、3、5、7d处死小鼠，无菌留取脾脏，免疫磁珠法分选CD4＋T细胞与CD4＋CD25＋T细胞（CD4＋CD25＋Trego流式细胞术检测CD4＋CD25＋Treg胞内叉头蛋白翼状螺旋转录因子P3（ Forkhead/winged helix transcription factor p3, Foxp3 ），CCK-8法检测脾效应T淋巴细胞增殖，实时定量PCR（Real—timePCR）检测Treg中miR-10a的基因表达水平。小鼠尾静脉注射慢病毒下调miR-10a，观察小鼠免疫功能。数据采用SPSS19．0统计软件进行数据处理，两组样本比较采用独立样本t检验，多组均数比较采用单因素方差分析（ANOVA）和Dunnett-t检验。结果正常组小鼠CD4＋CD25＋Treg细胞在CD4＋T细胞中的比例为（7．34±1．2）％，造模后脓毒症小鼠脾脏CD4＋CD25＋Treg比例1、3、5、7d明显升高（P〈0．05o与假手术组相比，脓毒症小鼠Foxp3的平均荧光强度（ mean fluorescence intensity, MFI ）也明显高于假手术组（P〈0．05）。Real-imePCR检测CD4＋CD25＋reg中miR-10a的表达。结果发现假手术组小鼠与正常组小鼠相比差异无统计学意义（P〉0．05）。毒症小鼠Treg中miR-0a表达较假手术组升高（P〈0．05o脓毒症鼠尾静脉注射携带miR-10a抑制序列的慢病毒后，CD4＋CD25＋Treg／CD4＋T比例明显升高（P〈0．05），且Foxp3的平均荧光强度也明显增强（P〈0．05），CD4＋T细胞的增殖能力相应减弱（P〈0．05）。结论在脓毒症致病过程Treg中miR-10a表达上调，抑制miR-10a水平能够显著提高Treg的比例和促进免疫抑制功能。鉴于CD4＋CD25＋Treg在脓毒症免疫功能紊乱中的重要作用，miR-10a可能是脓毒症免疫调理治疗的有效靶点。
Objective To investigate the role of miR-10a in CD4＋CD25＋Treg-mediated immunosuppression during sepsis and its potential role in immunotherapy for sepsis. Methods Sepsis mouse model was established by cecal ligation and puncture（CLP）. Balb/c mice of clean grade were sacrificed 1, 3, 5, and 7 days after operation. Blood as well as spleen samples were harvested at given intervals. The splenic CD4＋CD25＋Treg cells and CD4＋T cells were isolated by MACS microbeads. Cells were cultured, and phenotypes were analyzed by flow cytometry. The miR-10a expressed in Treg ceils were detected by Real-time PCR. After administration of LV-mmu-miR-10a-5p-inhibition, the immunosuppressive function have been detected. Statistical analyses were performed using one-way analysis of variance （SPSS19.0, Chicago, USA） test followed by Dunnett-t test to compare among three or more groups or by Student＇s t-test to compare between two groups. Results The percentages of splenic Tregs （CD4＋CD25＋/CD4＋T） was （7.34±1.2）% in normal group, and the increase in percentage of Tregs in spleen has been observed in septic mice （P〈0.05）. The mean fluorescence intensity （MFI） of Foxp3＋Treg was increased in septic mice compared with sham group （P〈0.05）. The expression of miR- 10a was significantly elevated on CLP 1-7 day （P〈0.05）. After down-regulation of miR-10a in septic mice, the percentages of Tregs （CD4＋CD25＋/CD4＋T） was significantly increased in septic mice （P〈0.05）, the MFI of Foxp3＋Treg was increased in septic mice compared with control group （P〈0.05）. The CD4＋T cell proliferative activity in CLP-induced mice was significantly suppressed on CLP 3 day compared with sham group （P〈0.05）.After down-regulation of miR-10a in septic mice, the CD4＋T cell proliferative activity was significantly suppressed compared with control group （P〈0.05）. Conclusions Treg plays a critical role in immunosuppression in septic mice. Inhibition of miR-10a in vivo could enhence immunesuppression of CD4＋CD25＋Treg. Therefore miR-10a may participate in the regulation of CD4＋CD25＋Treg inununosuppression in sepsis and become the target for immunotherapy.
出处
《中华急诊医学杂志》
CAS
CSCD
北大核心
2018年第2期152-158,共7页
Chinese Journal of Emergency Medicine
基金
国家自然科学基金（81401621、81571937）
浙江省自然科学基金（LY13H150004、LY13H150006、LY15H150006）
浙江省中医药优秀青年人才基金（2013ZQ023）
温州市科技计划项目（Y20150033）
