白细胞介素4调控巨噬细胞极化及骨髓间充质干细胞的成骨分化  

Interleukin-4 regulates macrophage polarization and osteogenic differentiation of bone marrow mesenchymal stem cells

作　　者：张洁 肖天骄 李丽 康佳兵 湛济帆 韦艳 田艾 Zhang Jie;Xiao Tianjiao;Li Li;Kang Jiabing;Zhan Jifan;Wei Yan;Tian Ai(First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine,Guiyang 550001,Guizhou Province,China;School of Stomatology,Guizhou Medical University,Guiyang 550001,Guizhou Province,China;Department of Restoration and Implant,Affiliated Stomatological Hospital of Guizhou Medical University,Guiyang 550001,Guizhou Province,China)

机构地区：[1]贵州中医药大学第一附属医院,贵州省贵阳市550001 [2]贵州医科大学口腔医学院,贵州省贵阳市550001 [3]贵州医科大学附属口腔医院修复种植科,贵州省贵阳市550001

出　　处：《中国组织工程研究》2024年第25期3960-3966,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目(81760192,82260193),项目负责人:田艾。

摘　　要：背景:白细胞介素4可以提高骨替代材料的成骨效应,但其分子机制尚未明确,进一步阐明白细胞介素4提高成骨效应的作用机制,有助于为牙槽骨缺损患者的再生治疗找到安全、经济和有效的方法。目的:探讨白细胞介素4对巨噬细胞极化转化和骨髓间充质干细胞成骨分化的影响及其可能的作用机制。方法:M1组RAW264.7细胞用干扰素γ+脂多糖诱导24 h,白细胞介素4+M1组RAW264.7细胞用干扰素γ+脂多糖诱导24 h后加入白细胞介素4诱导24 h,白细胞介素4+AG+M1组RAW264.7细胞用干扰素γ+脂多糖诱导细胞24 h后加入白细胞介素4和JAK/STAT通路抑制剂AG-490诱导24 h。干预结束后,采用免疫荧光染色分析巨噬细胞表型标记蛋白诱导型一氧化氮合酶、CD206的表达;采用ELISA试剂盒检测细胞培养上清液中白细胞介素10、肿瘤坏死因子α水平;采用RT-qPCR检测NLRP3、白细胞介素1β、caspase-1的mRNA表达;采用Western blot检测JAK1/p-JAK1、STAT6/p-STAT6、NLRP3、pro-IL-1β和pro-caspase-1通路蛋白的表达水平。随后将以上4组RAW264.7细胞与骨髓间充质干细胞transwell间接共培养24 h,然后进行碱性磷酸酶染色、茜素红染色,采用RT-qPCR检测碱性磷酸酶、Ⅰ型胶原和骨钙素的mRNA表达。结果与结论:①免疫荧光、ELISA检测结果显示白细胞介素4可促进M2样巨噬细胞CD206和白细胞介素10的表达,抑制诱导型一氧化氮合酶和肿瘤坏死因子α的表达;②RT-qPCR结果显示白细胞介素4可抑制NLRP3、白细胞介素1β、caspase-1 mRNA表达;③Western blot结果显示白细胞介素4可促进JAK1/p-JAK1、STAT6/p-STAT6和NLRP3蛋白表达明显增加;④与白细胞介素4+M1组RAW264.7细胞共培养的骨髓间充质干细胞的碱性磷酸酶染色、茜素红染色明显增强,碱性磷酸酶、Ⅰ型胶原和骨钙素的mRNA表达明显升高。结果表明,白细胞介素4可能通过上调JAK1/STAT6通路,抑制NLRP3活化,从而促进巨噬细胞从BACKGROUND:Interleukin-4 can promote the osteogenic effect of bone substitute materials,but its molecular mechanism is not yet clear.Further elucidating the mechanism of interleukin-4 promoting osteogenic effect can help find safe,economical,and effective methods for the regeneration treatment of alveolar bone defects in patients.OBJECTIVE:To explore the effect of interleukin-4 intervention on polarization transformation of macrophages and osteogenic differentiation of bone marrow mesenchymal stem cells and its possible mechanism.METHODS:RAW264.7 cells in the M1 group were induced with interferon gamma+lipopolysaccharide for 24 hours.RAW264.7 cells in the interleukin-4+M1 group were induced with interferon gamma+lipopolysaccharide for 24 hours and then interleukin-4 was added for 24 hours.RAW264.7 cells in the interleukin-4+AG+M1 group were induced with interferon gamma+lipopolysaccharide for 24 hours,and then interleukin-4 and AG-490,a JAK/STAT pathway inhibitor,were added for 24 hours.After intervention,immunofluorescence staining was used to analyze the expression of inducible nitric oxide synthase and CD206,the phenotypic marker protein of macrophages.ELISA kit was used to detect the expression of interleukin-10 and tumor necrosis factor-αin the supernatant of cell culture.The gene expressions of nodular receptor protein-3(NLRP3),interleukin-1β,and caspase-1 were detected by RT-qPCR.The expression levels of tyrosine protein kinase 1(JAK1)/phosphorylated tyrosine protein kinase 1(p-JAK1),signal transduction and transcription activator 6(STAT6)/phosphorylated signal transduction and transcription activator 6(p-STAT6),NLRP3,pro-interleukin-1βand pro-caspase-1 were detected by western blot assay.Then,RAW264.7 cells in the above four groups were indirectly co-cultured with bone marrow mesenchymal stem cells by transwell for 24 hours,followed by alkaline phosphatase staining and alizarin red staining.The mRNA expressions of alkaline phosphatase,collagen type I,and osteocalcin were detected by RT-qPCR.RESULTS AND

关 键 词：巨噬细胞 白细胞介素4 NLRP3炎性小体 JAK/STAT信号通路 成骨分化 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R783.1