中华急诊医学杂志  2018, Vol. 27 Issue (2): 152-158
microRNA-10a对脓毒症小鼠脾脏CD4+CD25+Treg免疫功能的影响
陈隆望, 邱俏檬, 连洁, 李海啸, 洪广亮, 卢中秋, 赵光举     
325000 浙江省温州,温州医科大学附属第一医院急诊医学中心
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 采用清洁级雄性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、7 d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+Treg)。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-time PCR)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SPSS 19.0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、7 d明显升高(P < 0.05)。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-time PCR检测CD4+CD25+Treg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)。脓毒症小鼠Treg中miR-10a表达较假手术组升高(P < 0.05)。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。
关键词: 调节性T细胞     脓毒症     microRNA-10a     叉头蛋白翼状螺旋转录因子P3     免疫功能     效应T细胞     细胞增殖     平均荧光强度    
The effects of miR-10a on the immune function of splenic CD4+CD25+ Treg cells in septic mice
Chen Longwang , Qiu Qiaomeng , Lian Jie , Li Haixiao , Hong Guangliang , Lu Zhongqiu , Zhao Guangju     
Emergency Medicine Department, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China
Abstract: 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 cells 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 immunosuppression in sepsis and become the target for immunotherapy.
Key words: Regulatory T cells     Sepsis     MicroRNA-10a     Forkhead/winged helix transcription factor p3     Immune function     Effector T cell     Proliferative activity     Mean fluorescence intensity    

脓毒症(sepsis)最新定义是宿主对感染的特异性反应导致的威胁生命的器官功能障碍[1]。CD4+CD25+Treg主要由胸腺中T细胞发育而来,具有免疫无能性和免疫抑制性两大特性[2]。前期实验及国外学者研究均证实,脓毒症免疫抑制状态与CD4+CD25+Treg密切相关[3-4]。miR-10a特异性表达于CD4+CD25+Treg,而在CD4+T和CD8+T细胞等初始T细胞中不表达或低表达,被认为是CD4+CD25+Treg的特征分子[5-7]。有研究发现,miR-10a对于CD4+CD25+Treg免疫功能具有重要影响[7-8]。然而,迄今为止在脓毒症中miR-10a的表达规律及其与CD4+CD25+Treg免疫抑制功能的关系尚未阐明。本项目拟探讨miR-10a与脓毒症CD4+CD25+Treg免疫功能的关系,分析调控miR-10a对脓毒症免疫功能紊乱的影响和机理,拟为脓毒症的免疫调控治疗提供新的靶点和依据。

1 材料与方法 1.1 材料与试剂

CD4+CD25+调节性T细胞分选盒子及MiniMACS磁性分离仪

(试剂编号: 5160711130,美天旎生物技术有限公司,格拉德巴赫,德国);异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记抗小鼠CD4(试剂编号: 4321795)、别藻蓝蛋白(Allophycocyanin,APC)标记抗小鼠CD25(试剂编号: 4300566)、藻红蛋白(P-phycoerythrin,PE)标记抗小鼠Foxp3(试剂编号: 4292088)、别藻蓝蛋白(Allophycocyanin,APC)标记抗小鼠Foxp3、藻红蛋白(P-phycoerythrin,PE)标记抗小鼠CD25(eBioscience, 圣地亚哥, CA);抗CD3/CD28抗体(eBioscience, 圣地亚哥, CA)(试剂编号: B240221和B236704);Cell Counting Kit-8(CCK-8试剂盒)(试剂编号: 9108日本同仁,日本);总RNA提取Trizol试剂盒(试剂编号: 9108,TaKaRa公司,大连,中国);逆转录-试剂盒(试剂编号: RR037A,TaKaRa公司,大连,中国);RealMasterMix(SYBRGreen)试剂盒(试剂编号: RR820A,TaKaRa公司,大连,中国);携带miR-10a抑制序列的慢病毒(吉凯生物有限公司,上海,中国)。

1.2 盲肠结扎穿孔(cecal ligation and puncture,CLP)脓毒症小鼠模型建立

雄性清洁级BALB/c小鼠,禁食24 h,自由饮水,1 d后腹腔注射1%戊巴比妥钠10 mg/kg麻醉后,固定小鼠在泡沫板上,75%酒精棉球消毒小鼠腹部手术区域。采用正中切口1.5~2 cm,眼科剪剪开,镊子钝性分离,剪开腹膜,找到盲肠并使其游离。于盲肠根部至盲肠远端的1/2~1/3处结扎盲肠,20号针头穿透结扎线远端盲肠的两侧肠壁,使肠内容物少量自穿刺孔溢出,将盲肠还纳回腹腔,用1-0丝线逐层单纯间断缝合缝合腹膜、筋膜和腹部肌肉关腹,术毕立即皮下注射37 ℃预热的1 mL生理盐水复苏;只开腹、牵引盲肠、复位、关腹、复苏,而里面盲肠既不结扎,也不穿孔的作为假手术组。手术完成后把小鼠仰放在盛有垫料的笼子内,添加食物与水,室内温度控制在(22±2)℃,湿度为40%~60%。每12 h昼夜交替一次,每6 h观测一次小鼠的状态。

脓毒症模型稳定性:本实验制作的小鼠CLP模型均符合国内外学者提出脓毒症动物模型的标准: (1)具有典型的实验室脓毒症表现如嗜睡、精神萎靡、反应迟钝、活动较少、眼角有分泌物、腹泻、竖毛等;(2)具有较高的脓毒症病死率满足72 h病死率在40%~60%的要求;(3)一般从造模后12 h出现死亡。本实验在严格控制实验条件下,使脓毒症小鼠达到较稳定的临床表现与病死率,以满足实验需要。

1.3 实验分组与给药

健康雄性清洁级BALB/c小鼠80只,体质量20~25 g,由上海斯莱克实验动物有限责任公司提供,生产许可证号: SCXK(沪)2012-0002。小鼠饲养及动物实验在温州医科大学实验动物中心进行,使用许可证号: SYXK (浙)2010.0150。实验遵循国际通行的动物福利和伦理准则。48只BALB/c小鼠随机分为6组:正常组、假手术组(sham)、CLP1天组、CLP3天组、CLP5天组、CLP7天组,分离CD4+CD25+Treg细胞,检测其免疫功能和miR-10a表达情况。根据实验结果,用miR-10a抑制序列的慢病毒(上海吉凯公司完成)干预,实验分为假手术组,CLP3天组,慢病毒阴性对照组(control组),miR-10a下调组(miR-10a-down组),每组8只小鼠,miR-10a下调组是在造模前24 h尾静脉注射滴度为4×107TU的300 μL慢病毒,对照组则相同时间点尾静脉注射300 μL阴性对照病毒,进一步检测小鼠免疫状态。

1.4 脓毒症小鼠脾脏CD4+CD25+Treg比例和Foxp3的表达检测

小鼠脾脏分出单个核细胞,制备细胞悬液,调整浓度为2×107/mL。染色之前加0.5 μg~ 1 μg/100 μL CD16/32抗体,孵育20 min。等分细胞悬液,每管50 μL,再补加50 μL染色液。加入适量抗体CD4-FITC 0.25 μL、CD25-APC 0.75 μL,四度避光孵育30 min。加2 mL流式染色液,洗涤,300 g离心弃上清液,重复一次。加1 mL 1×Foxp3固定和穿透工作液(Fixation/permeabilization),斡旋固定细胞。室温或四度避光孵育30~60 min。每管加2 mL通透缓冲液,300 g室温离心5 min, 弃上清液。加100 μL通透缓冲液重悬细胞后,加入FOXP3-PE 2.5μL,室温避光孵育20 ~30 min。然后用2 mL通透缓冲液洗一次,300 g室温离心5 min, 弃上清液。再加入2 mL流式染色液,300 g室温离心5 min, 弃上清液。300 μL流式染色液重悬,即可上机检测。

1.5 CD4+CD25+Treg和CD4+CD25-T细胞分离

麻醉后断颈处死小鼠,无菌取脾脏,淋巴细胞分离单个核细胞,根据细胞计数结果向细胞悬液中加入10 μL生物素鸡尾酒标记抗体(Biotin-antibody Cocktail)/107细胞,4 ℃,孵育10 min。每107个细胞加30 μL PBS和20 μL的Anti-Biotin Microbeads以及10 μL CD25-PE antibody,混匀细胞,4℃,孵育15 min。应用1~2 mL PBS/107细胞清洗细胞后1000 r/min离心10 min,去上清液后500 μL PBS/108细胞重悬,进行磁珠分选,阴选出未被标记的细胞就是CD4+T细胞。加入Anti-PE Microbeads 4℃避光孵育15 min,洗完后用PBS重悬后进行磁珠分选,阳选出标记的CD4+CD25+T细胞,未标记的为CD4+CD25-T细胞。流式细胞术鉴定CD4+CD25+Treg和CD4<