摘要
目的探讨小鼠实验性自身免疫性脑脊髓炎(EAE)模型中,外源性和内源性红细胞膜相关蛋白(ERMAP)通过白细胞介素6/信号转录及转录激活因子3(IL-6/STAT3)/维甲酸相关孤核受体γt/(ROR-γt)信号通路对辅助性T细胞17(Th17)细胞分化的影响。方法流式细胞术验证不同浓度ERMAP-Ig融合蛋白功能;琼脂糖凝胶电泳鉴定ERMAP基因敲除小鼠。流式细胞术检测ERMAP-Ig融合蛋白在体外对Th17细胞分化影响。40只6周龄普通C57BL/6小鼠随机分为2组建立EAE模型:对照(control)-Ig和ERMAP-Ig组,每组20只;记录临床评分;流式细胞术检测EAE小鼠体内Th17细胞分化情况。40只6周龄已鉴定的野生型和ERMAP基因敲除小鼠分为2组建立EAE模型:ERMAP^(+/+)和ERMAP^(-/-)组,每组20只。记录临床评分;脊髓HE和固蓝(LFB)染色并进行组织学半定量评分。流式细胞术检测IL-17A+CD4+T细胞百分比;Western blotting检测IL-6、白细胞介素17(IL-17)、STAT3、磷酸化STAT3(p-STAT3)、ROR-γt蛋白水平表达;Real-time PCR检测IL-17、肿瘤坏死因子α(TNF-α)、IL-6、STAT3和ROR-γt的mRNA表达;ELISA检测细胞水平IL-17和TNF-α的表达。结果1.外源性ERMAP-Ig融合蛋白抑制体内外Th17细胞分化且减轻小鼠EAE症状。2.与对照组相比,ERMAP^(-/-)组EAE小鼠炎性浸润和脱髓鞘症状加重,Th17分泌IL-17A增加。3.内源性ERMAP基因敲除后,IL-17、TNF-α、IL-6、STAT3及ROR-γt表达均增加。差异均有统计学意义(P<0.05)。结论ERMAP可能通过IL-6/STAT3/ROR-γt信号通路调控Th17细胞分化,参与小鼠EAE发生发展。
Objective To explore the effect of exogenous and endogenous erythrocyte membrane-associated protein(ERMAP)on helper T cell 17(Th17)cell differentiation through interleukin 6/signal transducers and activators of transcription 3/retionoid-related orphan nuclear receptor-γt(IL-6/STAT3/ROR-γt)signal pathway in the mouse model of experimental autoimmune encephalomyelitis(EAE).Methods Using flow cytometry to verify the function of ERMAP-Ig fusion protein at different concentrations;Agarose gel electrophoresis was performed to identify ERMAP knockout mice.Flow cytometry was performed to detect the effect of ERMAP-Ig fusion protein on Th17 cell differentiation in vitro.Forty 6-week-old normal C57BL/6 mice were randomly divided into 2 groups to establish EAE models,control-Ig and ERMAP-Ig groups,with 20 mice in each group;Clinical scores were recorded;Flow cytometry was performed to detect Th17 cell differentiation in EAE mice in vivo.Forty 6-week-old identified wild-type and ERMAP knockout mice were divided into 2 groups to establish EAE models.Identified wild-type and ERMAP knockout mice were divided into 2 groups to establish EAE models,ERMAP^(+/+)and ERMAP^(-/-)groups,with 20 mice in each group.Clinical scores were recorded;Spinal cord HE and luxol fast blue(LFB)staining and histological semi-quantitative scoring were performed.Flow cytometry was performed to detect the percentage of IL-17A+CD4+T cells;Western blotting was performed to detect the expression of IL-6,IL-17,STAT3,p-STAT3,and ROR-γt protein levels;Real-time PCR was performed to detect the mRNA expression of IL-17,TNF-α,IL-6,STAT3,and ROR-γt;ELISA was performed to detect the expression of IL-17 and TNF-αat the cellular level was detected by ELISA.Results 1.Exogenous ERMAP-Ig fusion protein inhibited Th17 cell differentiation and attenuated EAE symptoms in mice.2.Compared with the control group,ERMAP^(-/-)EAE mice showed increased inflammation and demyelination symptoms and increased Th17 secretion of IL-17A.3.Endogenous ERMAP knockdown resulted in increased expression of IL-17,TNF-α,IL-6 STAT3,and ROR-γt.All differences were statistically significant(P<0.05).Conclusion ERMAP may regulate Th17 cell differentiation through the IL-6/STAT3/ROR-γt signaling pathway and is involved in the development of EAE in mice.
作者
何可可
李远迪
文廷浩
朱婕
高杰
胡蓉
韩锋
苏敏
HE Ke-ke;LI Yuan-di;WEN Ting-hao;ZHU Jie;GAO Jie;HU Rong;HAN Feng;SU Min(Department of Histology and Embryology,Basic Medical College,Guizhou Medical University,Guiyang 550025,China;National Joint Local Engineering Laboratory for Cell Engineering and Biomedicine Technique,Guizhou Province Key Laboratory of Regenerative Medicine,Key Laboratory of Adult Stem Cell Translational Research(Chinese Academy of Medical Sciences),Guizhou Medical University,Guiyang 55025,China;Translational Medicine Research Center,Guizhou Medical University,Guiyang 55025,China;Department of Neurosurgery,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,China)
出处
《解剖学报》
CAS
CSCD
北大核心
2023年第5期538-545,共8页
Acta Anatomica Sinica
基金
国家自然科学基金(82171343,82160151)
贵州省科技计划项目(黔科合支撑[2020]4Y230号)
贵州省国际科技合作项目(黔科合平台人才[2020]4103号)
贵州省卫生健康委科学技术基金(gzwkj2022-086)。
