弗氏完全佐剂肌肉注射诱发炎性痛敏及免疫应答的转录组测序分析  

RNA-sequencing analysis of inflammatory pain hypersensitivity and immune response induced by complete Freund’s adjuvant intramuscular injection

作　　者：石惊涛 张晓宁[1] 曹婉萦 何伟[1] 刘一寒 王睿[2] 宿杨帅[1] 万红叶 曲正阳 景向红[1] 王晓宇[1] SHI Jingtao;ZHANG Xiaoning;CAO Wanying;HE Wei;LIU Yihan;WANG Rui;SU Yangshuai;WAN Hongye;QU Zhengyang;JING Xianghong;WANG Xiaoyu(Institute of Acupuncture and Moxibustion CACMS,Beijing 100700,China;Beijing Institute of Technology,Beijing 100081)

机构地区：[1]中国中医科学院针灸研究所,北京100700 [2]北京理工大学,北京100081

出　　处：《中国比较医学杂志》2022年第6期31-40,共10页Chinese Journal of Comparative Medicine

基　　金：科技部重点研发计划(2019YFC1709002);国家自然科学基金(81674083)。

摘　　要：目的观察弗氏完全佐剂(complete Freund’s adjuvant,CFA)肌肉注射后1~15 d的炎性反应,明确免疫应答反应的发生,通过分析转录组学的差异,为肌肉炎性反应及其诱发的痛觉敏化研究提供重要的参考。方法90只Wistar大鼠随机分为正常组和CFA注射后第1天(MD1)、第3天(MD3)、第5天(MD5)、第7天(MD7)、第9天(MD9)、第11天(MD11)、第13天(MD13)和第15天(MD15)模型组。在模型组大鼠左侧股二头肌肌腹注入200μL的CFA,观察各组大鼠左侧足底的机械痛阈值和热痛阈值变化;采用电化学发光法检测各组大鼠肌肉组织和外周血中炎性因子的含量;使用HE染色对正常组、MD3和MD13组大鼠注射侧局部肌肉组织进行形态学观察;采用基因组学的方法,分析这两组大鼠差异基因的表达。结果(1)股二头肌肌肉注射CFA可诱发同侧足底持续的机械痛敏和热痛敏。(2)CFA肌肉注射后24 h后,外周血和局部组织中IL-6、IL-1β和TNF-α的含量明显升高,局部组织中促炎因子含量在注射后第13天再次明显升高。(3)在CFA注射后第3天局部组织以中性粒细胞浸润为主,在CFA注射后第13天则以单核细胞浸润为主。(4)与MD3组相比,MD13组大鼠局部组织中与免疫反应相关的差异表达基因上调,其中涉及到Th细胞的分化、细胞因子受体相互作用等与适应性免疫相关的生物学通路。(5)CFA注射后局部组织中痛敏相关基因的表达水平升高,包括Ccl3、Cxcl1、Cxcl2、Cxcl9、Cxcl10等基因。结论CFA的注射可有效刺激固有免疫和适应性免疫的发生,诱导炎性痛敏,其中不同趋化因子家族基因在不同的免疫应答中发挥了重要的作用。Objective To observe 1~15 days of local inflammatory responses after an intramuscular injection of complete Freund’s adjuvant(CFA),clarify the result ing immune response,analyze transcriptomics differences amongst different immune responses,and provide a reference for studies of muscle inflammatory responses.Methods Ninety Wistar rats were randomized into control and model groups at day 1(MD1),3(MD3),5(MD5),7(MD7),9(MD9),11(MD11),13(MD13)and 15(MD15)after CFA injection after CFA injection.The 200μL of CFA was injected in the left femoral biceps abdomen of the mice in the model groups.The changes in mechanical pain and heat pain thresholds were observed in the left hindpaw of mice in each group.The levels of inflammatory factors were measured in the muscle tissue and peripheral blood of each group by electrochemiluminescence.Morphological observations of the local muscle tissue on the injection side of rats in the control,MD3 and MD13 groups were performed using HE staining.The differential expression of genes in the control and model groups was analyzed using genomics.Results(1)Intramuscular CFA injection induced sustained mechanical and heat hyperalgesia of the hindpaw.(2)At 24 h after CFA intramuscular injection,the levels of IL-6,IL-1βand TNF-αwere significantly elevated in the peripheral blood and local tissues,and the pro-inflammatory factor content in the local tissue was significantly elevated again at day 13 after CFA injection.(3)Local tissue was infiltrated mainly by neutrophils on day 3 after CFA injection and mainly by monocytes on day 13 after CFA injection.(4)The differentially expressed genes associated with the immune response are upregulated in local tissues in MD13 group as compared with their levels in MD3 group;These genes are involved in biological pathways related to adaptive immunity,such as Th-cell differentiation and cytokine-cytokine receptor interaction.(5)The expression levels of pain-related genes,including Ccl3,Cxcl1,Cxcl2,Cxcl9 and Cxcl10,were elevated in local tissues after CF

关 键 词：弗氏完全佐剂 肌肉炎性痛 细胞因子 固有免疫反应 适应性免疫反应 转录组测序 

分 类 号：R-33[医药卫生]