脱细胞软骨基质对巨噬细胞极化的调控  

Regulatory effect of decellularized cartilage matrix on macrophage polarization

作　　者：田广招 杨振 查康康[1,2,3] 孙志强 李旭 眭翔[2] 黄靖香[2] 郭全义[2] 刘舒云[2] Tian Guangzhao;Yang Zhen;Zha Kangkang;Sun Zhiqiang;Li Xu;Sui Xiang;Huang Jingxiang;Guo Quanyi;Liu Shuyun(PLA Medical College,Beijing 100853,China;Institute of Orthopedics,First Medical Center,Chinese PLA General Hospital,Beijing Key Laboratory of Regenerative Medicine in Orthopedics,Key Laboratory of Musculoskeletal Trauma&War Injuries,PLA,Beijing 100853,China;Medical College of Nankai University,Tianjin 300071,China)

机构地区：[1]解放军医学院,北京市100853 [2]中国人民解放军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]南开大学医学院,天津市300071

出　　处：《中国组织工程研究》2021年第22期3545-3550,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家重点研发计划课题(2018YFC1105900),项目参与者:刘舒云。

摘　　要：背景:细胞外基质来源的生物材料在组织工程再生领域应用广泛,近年来生物材料对巨噬细胞表型极化作用受到极大关注。目的:探究脱细胞软骨基质(decellularized cartilage extracellular matrix,DCM)及胃蛋白酶处理脱细胞软骨基质(pepsin treated decellularized cartilage extracellular matrix,PDCM)对巨噬细胞表型极化的调控作用。方法:采用物理粉碎、反复冻融及差速离心方法制备猪关节软骨脱细胞软骨基质(DCM),溶解于胃蛋白酶溶液内得到其生物降解产物(PDCM)。将小鼠巨噬细胞系RAW264.7分5组培养:对照组加入巨噬细胞完全培养基,M1型阳性对照组加入含脂多糖+干扰素γ的完全培养基,M2型阳性对照组加入含白细胞介素4的完全培养基,PDCM组加入含PDCM的完全培养基,胃蛋白酶组加入含胃蛋白酶的完全培养基,培养18 h后进行CD86(M1型巨噬细胞)、CD206(M2型巨噬细胞)免疫荧光染色鉴定。另外将小鼠巨噬细胞系RAW264.7分6组培养:对照组、M1型阳性对照组、M2型阳性对照组、胃蛋白酶组、DCM涂布组与PDCM涂布组,培养24 h后进行CD86、CD206流式细胞术检测。结果与结论:①免疫荧光结果显示,PDCM组诱导巨噬细胞CD86表达阳性比约为0.13,诱导巨噬细胞CD206表达阳性比约为0.5;②流式细胞术检测结果显示,DCM诱导巨噬细胞表达CD86(20.1%)及CD206(28.8%),PDCM只诱导巨噬细胞表达CD206(23.2%);③结果表明,DCM及其生物降解产物PDCM具有促进巨噬细胞M2型极化的作用。BACKGROUND:Biomaterials derived from extracellular matrix have been successfully applied in the field of tissue engineering regeneration.In recent years,great attention has been paid to the effect of biomaterials on the phenotypic polarization of macrophages.OBJECTIVE:To investigate the regulatory effects of decellularized cartilage extracellular matrix(DCM)and pepsin-treated decellularized cartilage extracellular matrix(PDCM)on phenotypic polarization of macrophages.METHODS:The DCM of the pig was prepared by physical comminution,repeated freeze-thaw and differential centrifugation.The biodegradation product(PDCM)was obtained by dissolving it in pepsin solution.Mouse macrophage cell line RAW264.7 was divided into five groups.In the control group,complete macrophage culture medium was added.In the M1 positive control group,lipopolysaccharide+interferonγsolution was added.In the M2 positive control group,interleukin 4 solution was added.In the PDCM group,PDCM solution was added.In the pepsin group,pepsin solution was added.After 18 hours of culture,immunofluorescence staining for CD86(M1 type macrophages)and CD206(M2 type macrophages)was performed.In addition,mouse macrophage cell line RAW264.7 was divided into six groups:control group,M1 positive control group,M2 positive control group,pepsin group,DCM coating group and PDCM coating group.After 24 hours of culture,CD86 and CD206 were detected by flow cytometry.RESULTS AND CONCLUSION:(1)Immunofluorescence results showed that in the PDCM group,induced CD86 expression ratio was approximately 0.13;induced CD206 expression ratio was approximately 0.5.(2)Flow cytometry results showed that DCM induced CD86(20.1%)and CD206(28.8%)expression in macrophages;PDCM only induced CD206 expression(23.2%).(3)The results showed that DCM and its biodegradation products could promote M2 polarization of macrophages.

关 键 词：软骨 材料 组织工程 脱细胞 软骨基质 巨噬细胞极化 巨噬细胞表型 

分 类 号：R459.9[医药卫生—治疗学] R318.8[医药卫生—临床医学]