摘要
背景:巨噬细胞极化介导的炎症反应在骨关节炎、类风湿关节炎等慢性炎症性疾病中发挥着重要作用。磷酸肌酸改性壳聚糖水凝胶在组织修复中表现出良好的潜在应用前景,但该水凝胶对巨噬细胞极化与炎症因子表达的作用尚不清楚。目的:探究磷酸肌酸改性甲基丙烯酸酐化壳聚糖水凝胶对巨噬细胞极化和炎症因子表达的影响。方法:采用一步冻干法制备水溶性良好的磷酸肌酸改性甲基丙烯酸酐化壳聚糖,在低毒性引发剂及紫外光照下进一步制备水凝胶。提取SD大鼠骨髓源性巨噬细胞,培养7 d后分3组培养:对照组加入细胞完全培养基;脂多糖组加入含脂多糖的细胞完全培养基;壳聚糖水凝胶组加入冻干水凝胶样品与含脂多糖的细胞完全培养基。利用CCK-8法检测细胞活性,RT-PCR、ELISA、Western Blot和免疫荧光技术检测巨噬细胞极化及其炎症因子表达情况。结果与结论:①CCK-8实验检测显示,培养1,3,5 d后,3组细胞活性比较差异无显著性意义(P>0.05);②Western Blot和免疫荧光染色结果表明,与对照组比较,脂多糖组培养1,3 d的M2型巨噬细胞表面标志物CD206蛋白表达下调(P<0.05),ARG1蛋白表达上调(P<0.05);与脂多糖组比较,壳聚糖水凝胶组培养3 d的CD206、ARG1蛋白表达均上调(P<0.05);③ELISA与RT-PCR检测结果显示,与对照组相比,脂多糖组培养1,3 d促炎因子白细胞介素1β、白细胞介素6的蛋白与mRNA表达增加(P<0.05);与脂多糖组比较,壳聚糖水凝胶组培养1,3 d白细胞介素1β、白细胞介素6的蛋白与mRNA表达均减少(P<0.05);④结果表明,磷酸肌酸改性甲基丙烯酸酐化壳聚糖水凝胶促进巨噬细胞M2型极化、抑制其促炎因子表达的作用,将来可能为免疫相关疾病提供潜在治疗手段。
BACKGROUND:Macrophage polarization-mediated inflammation plays an important role in chronic inflammatory diseases such as osteoarthritis and rheumatoid arthritis.Phosphocreatine-modified chitosan hydrogels show good potential application prospects in tissue repair,but the effect of this new hydrogel on polarization and inflammatory factor expression in macrophages is still unclear.OBJECTIVE:To explore the effect of phosphocreatine modified methacrylic anhydride chitosan hydrogel on macrophage polarization and inflammatory factor expression.METHODS:One-step freeze-drying method was used to prepare phosphocreatine modified methacrylic anhydride chitosan with good water solubility.Hydrogels were further prepared under low toxicity initiators and ultraviolet irradiation.Bone marrow-derived macrophages were extracted from SD rats and cultured for 7 days,and then divided into three groups.Complete cell culture medium was added in the control group.Complete cell culture medium containing lipopolysaccharide was added in the lipopolysaccharide group.Freeze-dried hydrogel samples and cell complete medium containing lipopolysaccharide were added in the chitosan hydrogel group.Cell viability was detected using CCK-8 assay.RT-PCR,ELISA,western blot assay,and immunofluorescence staining were used to detect macrophage polarization and the expression of inflammatory factors.RESULTS AND CONCLUSION:(1)CCK-8 assay showed that after 1,3,and 5 days of culture,there was no significant difference in the cell viability among the three groups(P>0.05).(2)Western blot assay and immunofluorescence staining showed that compared with the control group,the M2 macrophage surface marker CD206 protein expression was down-regulated(P<0.05)and the ARG1 protein expression was up-regulated(P<0.05)in the lipopolysaccharide group after 1 and 3 days of culture.Compared with the lipopolysaccharide group,the protein expression levels of CD206 and ARG1 in the chitosan hydrogel group were up-regulated after 3 days of culture(P<0.05).(3)ELISA and RT-PCR resu
作者
生卫北
熊奡
刘苏
邓嘉鹏
翁鉴
于斐
陈英奇
曾晖
Sheng Weibei;Xiong Ao;Liu Su;Deng Jiapeng;Weng Jian;Yu Fei;Chen Yingqi;Zeng Hui(Weifang Medical University,Weifang 261053,Shandong Province,China;Department of Bone&Joint Surgery,Shenzhen Hospital of Peking University,Shenzhen 518036,Guangdong Province,China;National&Local Joint Engineering Research Center for Orthopedic Biomaterials,Shenzhen 518036,Guangdong Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2022年第31期5040-5046,共7页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金(82172432),项目负责人:曾晖
国家自然科学基金(82102568),项目负责人:于斐
国家自然科学基金(82001319),项目负责人:翁鉴
广东省基础与应用基础研究基金(2019A1515011290),项目负责人:曾晖
骨科生物材料国家地方联合工程研究中心(XMHT20190204007),项目负责人:曾晖
深圳市高水平医院建设基金(SZXK023),项目负责人:曾晖
深圳市“三名”医学项目(SZSM201612092),项目负责人:曾晖
深圳市研发项目(Z2021N054),项目负责人:曾晖。
关键词
壳聚糖水凝胶
骨髓源性巨噬细胞
极化
炎症因子
脂多糖
chitosan hydrogel
bone marrow-derived macrophage
polarization
inflammatory factor
lipopolysaccharide
