中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (31): 5040-5046.doi: 10.12307/2022.959

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

磷酸肌酸改性壳聚糖水凝胶干预大鼠骨髓源性巨噬细胞极化和炎症因子的表达

生卫北1,2,3,熊 奡2,3,刘 苏2,3,邓嘉鹏2,3,翁 鉴2,3,于 斐2,3,陈英奇2,3,曾 晖2,3   

  1. 1潍坊医学院,山东省潍坊市   261053;2北京大学深圳医院骨关节科,广东省深圳市   518036;3骨科生物材料国家地方联合工程研究中心,广东省深圳市   518036
  • 收稿日期:2021-12-03 接受日期:2022-01-17 出版日期:2022-11-08 发布日期:2022-04-25
  • 通讯作者: 曾晖,主任医师,教授,北京大学深圳医院骨关节科,广东省深圳市 518036;骨科生物材料国家地方联合工程研究中心,广东省深圳市 518036
  • 作者简介:生卫北,男,1994年生,河南省安阳市人,汉族,主要从事骨组织损伤修复与免疫调节研究。
  • 基金资助:
    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

Effect of phosphocreatine modified chitosan hydrogel on polarization and inflammatory factor expression in rat bone marrow-derived macrophages

Sheng Weibei1, 2, 3, Xiong Ao2, 3, Liu Su2, 3, Deng Jiapeng2, 3, Weng Jian2, 3, Yu Fei2, 3, Chen Yingqi2, 3, Zeng Hui2, 3   

  1. 1Weifang Medical University, Weifang 261053, Shandong Province, China; 2Department of Bone & Joint Surgery, Shenzhen Hospital of Peking University, Shenzhen 518036, Guangdong Province, China; 3National & Local Joint Engineering Research Center for Orthopedic Biomaterials, Shenzhen 518036, Guangdong Province, China
  • Received:2021-12-03 Accepted:2022-01-17 Online:2022-11-08 Published:2022-04-25
  • Contact: Zeng Hui, Chief physician, Professor, 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
  • About author:Sheng Weibei, 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
  • Supported by:
    National Natural Science Foundation of China, No. 82172432 (to ZH); National Natural Science Foundation of China, No. 82102568 (to YF); National Natural Science Foundation of China, No. 82001319 (to WJ); Guangdong Province Fundamental and Applied Basic Research Fund, No. 2019A1515011290 (to ZH); National and Local Joint Engineering Research Center for Orthopedic Biomaterials, No. XMHT20190204007 (to ZH); Shenzhen High-level Hospital Construction Fund, No. SZXK023 (to ZH); Shenzhen “Three Names” Medical Project, No. SZSM201612092 (to ZH); Shenzhen Research & Development Project, No. Z2021N054 (to ZH)

摘要:

文题释义:
水凝胶:是由水含量丰富的亲水聚合物组成的三维网络结构凝胶材料,有含水量高及具备多孔结构特点,利于细胞的黏附、增殖及营养物质与代谢产物的传输。
壳聚糖:通常从甲壳类动物的壳上提取而来,是一种线性天然多糖,在结构上与糖胺聚糖相似,具有良好的生物相容性、生物安全性、低毒性和低免疫刺激性,常用于骨/软骨组织工程支架的基质材料,如常用于水凝胶的合成。

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。

https://orcid.org/0000-0002-0716-5938 (生卫北)

中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程

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

Abstract: 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 results showed that compared with the control group, the protein and mRNA expression levels of pro-inflammatory factors interleukin-1β and interleukin-6 in the lipopolysaccharide group increased after 1 and 3 days of culture (P < 0.05). Compared with the lipopolysaccharide group, the protein and mRNA expression levels of interleukin-1β and interleukin-6 in the chitosan hydrogel group were decreased after 1 and 3 days of culture (P < 0.05). (4) These results have shown that phosphocreatine modified methacrylic anhydride chitosan hydrogel promotes M2-type polarization of macrophages and inhibits the expression of pro-inflammatory factors, which may provide a potential treatment for immune-related diseases in the future.

Key words: chitosan hydrogel, bone marrow-derived macrophage, polarization, inflammatory factor, lipopolysaccharide

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