脱细胞软骨细胞外基质对小鼠巨噬细胞系表型的调控  

Regulatory effect of acellular cartilage extracellular matrix on phenotype of mouse macrophage line

作　　者：李珺琦 田广招 陈明学 王皓 刘舒云 眭翔 黄靖香 李明[3] 郭全义 Li Junqi;Tian Guangzhao;Chen Mingxue;Wang Hao;Liu Shuyun;Sui Xiang;Huang Jingxiang;Li Ming;Guo Quanyi(Department of Basic Medicine,Changzhi Medical College,Changzhi 046000,Shanxi Province,China;Institute of Orthopedics of First Medical Center of Chinese PLA General Hospital,Beijing Key Laboratory of Orthopedic Regenerative Medicine,Key Laboratory of Military Orthopedic Warfare Trauma,Beijing 100853,China;Department of Anatomy,Changzhi Medical College,Changzhi 046000,Shanxi Province,China)

机构地区：[1]长治医学院基础医学部,山西省长治市046000 [2]中国人民解放军总医院第一医学中心骨科研究所,骨科再生医学北京市重点实验室,全军骨科战创伤重点实验室,北京市100853 [3]长治医学院解剖教研室,山西省长治市046000

出　　处：《中国组织工程研究》2021年第10期1512-1516,共5页Chinese Journal of Tissue Engineering Research

基　　金：国家重点研发计划项目(2018YFC1105900),项目参与者:刘舒云;国家自然科学基金委员会资助项目(81772319),项目负责人:郭全义。

摘　　要：背景:前期研究证实脱细胞软骨细胞外基质可有效促进关节软骨缺损修复再生,具有良好的修复效果,但是其促再生机制尚未阐明。目的:探究猪来源脱细胞软骨细胞外基质支架对小鼠巨噬细胞系表型极化的调控。方法:以猪膝关节软骨为原料,通过湿法粉碎、差速离心、冷冻干燥的方法制备脱细胞软骨细胞外基质支架。将小鼠RAW264.7巨噬细胞系接种于脱细胞软骨细胞外基质支架,构建细胞-支架复合物,体外极化诱导4 d,采用扫描电镜观察细胞黏附情况,死/活细胞染色观察细胞生长情况,免疫荧光染色观察M1型巨噬细胞特征性表面标志物CD86和M2型巨噬细胞特征性表面标志物CD206的表达,进而明确极化诱导情况。结果与结论:①扫描电镜显示,复合物中的巨噬细胞为原来的圆形或类圆形,沿着支架的管状排列,广泛分布在支架表面及内部结构中;②死/活细胞染色显示,支架上附着的大部分细胞为活细胞,仅有极少数死细胞;③免疫荧光染色显示,复合物中的M2型巨噬细胞标志物CD206呈80%的高阳性表达,M1型巨噬细胞标志物CD86的阳性表达甚少;④结果表明,猪来源脱细胞软骨细胞外基质支架可诱导极化M0型巨噬细胞为修复性M2型巨噬细胞。BACKGROUND: Previous studies have confirmed that the extracellular matrix of acellular cartilage can effectively promote the repair and regeneration of articular cartilage defects, and has a good repair effect, but its mechanism of promoting regeneration has not been elucidated.OBJECTIVE: To investigate the regulation of phenotypic polarization of mouse macrophage cell line by extracellular matrix scaffolds derived from pigs.METHODS: Using pig knee articular cartilage as raw material, the decellularized cartilage extracellular matrix scaffold was prepared by wet grinding, differential centrifugation, and freeze drying. The mouse RAW264.7 macrophage cell line was inoculated on the decellularized cartilage extracellular matrix scaffold to construct a cell-scaffold complex, which induced polarization in vitro for 4 days. Cell adhesion was observed using scanning electron microscopy, and cell growth was observed with dead/viable cells. Immunofluorescence staining was used to observe the expression of CD86, a characteristic surface marker of M1-type macrophages, and CD206, a characteristic surface marker of M2-type macrophages, and to clarify the polarization induction.RESULTS AND CONCLUSION:(1) Scanning electron microscopy showed that the macrophages in the complex were originally round or quasi round, arranged along the tube of the scaffold, and widely distributed on the surface and internal structure of the scaffold.(2) Dead/live cell staining showed that most of the cells attached to the scaffold were living cells, with only a few dead cells.(3) Immunofluorescence staining showed that the M2 type macrophage marker CD206 in the complex was 80% highly expressed. The positive expression of CD86 the marker of M1 type macrophages was scarce.(4) The results show that the porcine-derived acellular cartilage extracellular matrix scaffold can induce polarized M0 type macrophages to repair M2 type macrophages.

关 键 词：骨 软骨 再生 细胞外基质 巨噬细胞 极化 组织工程 免疫 

分 类 号：R459.9[医药卫生—治疗学] R392[医药卫生—临床医学]