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石惊涛,张晓宁,曹婉萦,何伟,刘一寒,王睿,宿杨帅,万红叶,曲正阳,景向红,王晓宇.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[J].中国比较医学杂志,2022,32(6):31~40.

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

投稿时间：2021-09-02

DOI：10. 3969 / j.issn.1671-7856. 2022. 06. 005

中文关键词: 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

英文关键词:complete Freund ’ s adjuvant muscle inflammatory pain cytokines innate immune response adaptive immune response RNA-sequencing

基金项目:

作者	单位	E-mail
石惊涛	中国中医科学院针灸研究所,北京 100700	909208703@ qq. com
张晓宁	中国中医科学院针灸研究所,北京 100700
曹婉萦	中国中医科学院针灸研究所,北京 100700
何伟	中国中医科学院针灸研究所,北京 100700
刘一寒	中国中医科学院针灸研究所,北京 100700
王睿	北京理工大学,北京 100081
宿杨帅	中国中医科学院针灸研究所,北京 100700
万红叶	中国中医科学院针灸研究所,北京 100700
曲正阳	中国中医科学院针灸研究所,北京 100700
景向红	中国中医科学院针灸研究所,北京 100700
王晓宇	中国中医科学院针灸研究所,北京 100700	xiaorain_wang@ hotmailcom

Author Name	Affiliation	E-mail
SHI Jingtao	Institute of Acupuncture and Moxibustion CACMS, Beijing 100700, China	909208703@ qq. com
ZHANG Xiaoning	Institute of Acupuncture and Moxibustion CACMS, Beijing 100700, China
CAO Wanying	Institute of Acupuncture and Moxibustion CACMS, Beijing 100700, China
HE Wei	Institute of Acupuncture and Moxibustion CACMS, Beijing 100700, China
LIU Yihan	Institute of Acupuncture and Moxibustion CACMS, Beijing 100700, China
WANG Rui	Beijing Institute of Technology, Beijing 100081
SU Yangshuai	Institute of Acupuncture and Moxibustion CACMS, Beijing 100700, China
WAN Hongye	Institute of Acupuncture and Moxibustion CACMS, Beijing 100700, China
QU Zhengyang	Institute of Acupuncture and Moxibustion CACMS, Beijing 100700, China
JING Xianghong	Institute of Acupuncture and Moxibustion CACMS, Beijing 100700, China
WANG Xiaoyu	Institute of Acupuncture and Moxibustion CACMS, Beijing 100700, China	xiaorain_wang@ hotmailcom

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中文摘要:

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 200 μL 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。

英文摘要:

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 200 μL 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-1β and TNF-α were 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 CFA injection. Conclusions An injection of CFA can effectively stimulate innate and adaptive immune responses and induce inflammatory pain sensitization, in which different chemokine family genes play important roles in the different immune responses.

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