骨骼肌纤维NLRP3炎症小体激活、亚细胞定位及3D重建  

Activation,subcellular localization,and 3D reconstruction of NLRP3 inflammasome in skeletal muscle fibers

作　　者：厉洋洋 菅晓婷 黄静雯 王琪森 桂炜超 张小龙 赵子炜 胡稷杰[2] 廖华[1] Li Yangyang;Jian Xiaoting;Huang Jingwen;Wang Qisen;Gui Weichao;Zhang Xiaolong;Zhao Ziwei;Hu Jijie;Liao Hua(Guangdong Provincial Key Laboratory of Tissue Construction and Testing,Department of Anatomy,School of Basic Medical Sciences,Southern Medical University,Guangzhou 510515,China;Department of Orthopaedics and Traumatology,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China)

机构地区：[1]南方医科大学基础医学院解剖教研室/广东省组织构建与检测重点实验室,广东广州510515 [2]南方医科大学南方医院创伤骨科,广东广州510515

出　　处：《中国临床解剖学杂志》2025年第2期168-174,共7页Chinese Journal of Clinical Anatomy

基　　金：国家自然科学基金面上项目(32071181);广东省自然科学基金面上项目(2023A1515012191);国家重点研发计划(2022YFF1202603)。

摘　　要：目的探讨体外炎性环境中肌纤维内NLRP3炎症小体的激活、亚细胞定位及其3D重建。方法C2C12细胞体外培养、马血清分化;脂多糖(Lipopolysaccharide/LPS)及尼日利亚菌素(Nigericin/Nig)刺激C2C12肌管内NLRP3炎症小体激活;qPCR及Western blot分析NLRP3、ASC、Caspase-1基因及蛋白水平;免疫荧光分析NLRP3、ASC的聚集与共定位;共聚焦观察、Imaris软件重建显示聚合态NLRP3、ASC与特定细胞器(高尔基体、线粒体、内质网)的关联。结果体外条件下,LPS/Nig联合刺激可致肌纤维内NLRP3、ASC、Caspase-1基因、蛋白水平上调,胞浆内可见NLRP3、ASC聚集;肌纤维内线粒体功能分子TOM20、高尔基体分子TGN38显著上调,聚集态NLRP3、ASC与线粒体、高尔基体存在共定位。结论LPS/Nig刺激可诱导骨骼肌纤维内NLRP3炎症小体激活。活化的NLRP3炎症小体组分与线粒体及高尔基体密切关联。Objective To investigate the activation,subcellular localization,and 3D reconstruction of the NLRP3 inflammasome within muscle fibers under in vitro inflammatory conditions.Methods C2C12 cells were cultured in vitro and differentiated with horse serum.Lipopolysaccharide(LPS)and Nigericin(Nig)were used to stimulate NLRP3 inflammasome activation in C2C12 myotubes.qPCR and Western blot were employed to analyze the gene and protein levels of NLRP3,ASC,and Caspase-1.Immunofluorescence analysis was performed to assess NLRP3 and ASC aggregation and co-localization.Confocal microscopy and Imaris software were used to visualize and reconstruct the associations of aggregated NLRP3 and ASC with specific organelles,such as the Golgi apparatus,mitochondria,and endoplasmic reticulum.Results In vitro,LPS/Nig co-stimulation increased the gene and protein expression of NLRP3,ASC,and Caspase-1 in muscle fibers,with cytoplasmic aggregation of NLRP3 and ASC observed.The mitochondrial functional molecule TOM20 and Golgi marker TGN38 were significantly upregulated,and co-localization was detected between aggregated NLRP3,ASC,and both mitochondria and the Golgi apparatus.Conclusions LPS/Nig stimulation induces activation of the NLRP3 inflammasome in skeletal muscle fibers.The activated components of the NLRP3 inflammasome are closely associated with mitochondria and the Golgi apparatus.

关 键 词：NLRP3炎症小体 C2C12细胞 Imaris 3D重建 细胞器 

分 类 号：R392.31[医药卫生—免疫学]