中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (32): 5126-5132.doi: 10.12307/2023.590

• 软骨组织构建 cartilage tissue construction • 上一篇    下一篇

建立软骨细胞铁过载模型及损伤机制

何  琪1，2，潘兆丰1，2，陈柏豪1，2，杨均政1，2，黎少聪1，2，曾嘉旭1，2，周  驰2，3，王海彬2，3   

  1. 1广州中医药大学第一临床医学院，广东省广州市  510405；2广州中医药大学岭南医学研究中心，广东省广州市  510405；3广州中医药大学第一附属医院，广东省广州市  510405
  • 收稿日期:2022-08-02 接受日期:2022-09-18 出版日期:2023-11-18 发布日期:2023-03-23
  • 通讯作者: 王海彬，博士，主任医师，广州中医药大学岭南医学研究中心，广东省广州市 510405；广州中医药大学第一附属医院，广东省广州市 510405 周驰，博士，副主任医师，广州中医药大学岭南医学研究中心，广东省广州市 510405；广州中医药大学第一附属医院，广东省广州市 510405
  • 作者简介:何琪，男，1997年生，广东省揭阳市人，汉族，广州中医药大学在读博士，主要从事关节炎和骨质疏松的基础研究。
  • 基金资助:
    国家自然科学基金资助项目(82074462)，项目负责人：王海彬；广州中医药大学“双一流”与高水平大学学科协同创新团队项目(2021xk53)，项目负责人：王海彬

Establishment of chondrocyte model of iron overload and the mechanism of injury

He Qi1, 2, Pan Zhaofeng1, 2, Chen Baihao1, 2, Yang Junzheng1, 2, Li Shaocong1, 2, Zeng Jiaxu1, 2, Zhou Chi2, 3, Wang Haibin2, 3   

  1. 1First School of Clinical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; 2Lingnan Medical Research Center, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; 3First Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China
  • Received:2022-08-02 Accepted:2022-09-18 Online:2023-11-18 Published:2023-03-23
  • Contact: Wang Haibin, MD, Chief physician, Lingnan Medical Research Center, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; First Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China Zhou Chi, MD, Associate chief physician, Lingnan Medical Research Center, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; First Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China
  • About author:He Qi, MD candidate, First School of Clinical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; Lingnan Medical Research Center, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China
  • Supported by:
    National Natural Science Foundation of China, No. 82074462 (to WHB); “Double First-class” and High-level Collaborative Innovation Team Project of Guangzhou University of Chinese Medicine, No. 2021xk53 (to WHB)

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摘要：


文题释义：
铁过载：是由于人体缺乏排出多余铁的有效途径，衰老、基因突变、膳食铁摄入、慢性输血等诸多危险因素会导致机体进行性和病理性铁超负荷，引起器官、细胞的广泛受损。
氧化应激：是指体内氧化与抗氧化作用失衡的一种状态，也是自由基在体内产生的一种负面作用，被认为是导致衰老和疾病的一个重要因素。

背景：目前尚不清楚过量的铁是如何参与关节炎发病的，研究铁过载参与关节炎发展的潜在机制可以为关节炎的治疗提供新的方向。
目的：通过枸橼酸铁铵和软骨细胞共培养构建软骨细胞的铁过载模型，观察在铁过载环境下对软骨细胞存活、铁沉积、氧化应激以及线粒体损伤的影响。
方法：采用酶消化法提取C57BL/6J小鼠膝关节软骨细胞，加入不同浓度(100，250，500，1 000，2 000 μmol/L)的枸橼酸铁铵干预软骨细胞，分别培养24，48，72 h，建立铁过载软骨细胞模型，采用CCK-8法分析细胞活力变化，钙黄绿素AM法测定细胞内铁含量，筛选出后续实验组所用枸橼酸铁铵浓度；采用荧光探针DCFH-DA染色检测活性氧水平，以JC-1为荧光探针检测线粒体膜电位变化，并通过实时定量荧光PCR和Western blot检测Ⅱ型胶原、基质金属蛋白酶3和13的表达水平。
结果与结论：500和1 000 μmol/L浓度枸橼酸铁铵与软骨细胞共培养48 h可成功建立软骨细胞铁过载模型。枸橼酸铁铵能够诱导软骨细胞内铁过载，且铁过载会破坏线粒体功能，增加细胞内活性氧的水平，软骨细胞的功能活性受损，分泌Ⅱ型胶原、聚集蛋白聚糖等基质成分的能力下降，导致关节软骨自身修复难度进一步加大，这为今后进一步研究铁过载对软骨细胞的损伤机制以及探索治疗铁过载性关节炎的有效治疗药物奠定基础。
https://orcid.org/0000-0003-2753-2536(何琪)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 铁过载, 软骨细胞, 氧化应激, 线粒体功能

Abstract: BACKGROUND:  It is unclear how excessive iron is involved in the development of arthritis, and investigating the potential mechanisms by which iron overload is involved in the development of arthritis could provide new directions for the treatment of arthritis.
OBJECTIVE: To establish an iron overload model of chondrocytes by co-culture of ferric ammonium citrate and chondrocytes, and to observe the effects of iron overload on chondrocyte survival, iron deposition, oxidative stress, and mitochondrial damage. 
METHODS: The chondrocytes of C57BL/6J mouse knee joint were extracted by enzyme digestion and cultured with ferric ammonium citrate (100, 250, 500, 1 000 and 2 000 μmol/L) chondrocytes for 24, 48 and 72 hours to establish chondrocyte models of iron overload. Cell viability was measured by cell counting kit-8. Content of iron was determined by Calcein-AM, and ferric ammonium citrate concentration was screened for subsequent experiments. Then, the level of reactive oxygen species was detected by fluorescence probe DCFH-DA and the change of mitochondrial membrane potential was detected by JC-1. Real-time quantitative PCR and western blot assays were used to detect the expression levels of type II collagen, matrix metalloproteinases 3 and 13. 
RESULTS AND CONCLUSION: The iron overload model could be successfully established in chondrocytes co-cultured with ferric ammonium citrate at the concentration of 500 and 1 000 μmol/L for 48 hours. Ferric ammonium citrate could induce iron overload in chondrocytes, and iron overload subsequently destroy mitochondrial function, increase the level of intracellular reactive oxygen species, impair the functional activity of chondrocytes, and decrease the cell ability to secrete type II collagen and aggrecan, which further increases the difficulty of articular cartilage self-repair. It lays a foundation for further research on the mechanism of chondrocyte injury caused by iron overload and the exploration of effective therapeutic drugs for the treatment of iron overload-induced arthritis.

Key words: iron overload, chondrocyte, oxidative stress, mitochondrial function

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