中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (2): 223-229.doi: 10.12307/2022.914

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

循环牵张力作用下髓核外泌体对终板软骨细胞的影响

张伟业1,展嘉文1,2,朱立国2,3,王尚全1,2,陈  明1,2,魏  戌2,4,冯敏山3,4,于  杰3,4,韩  涛1,4,蔡楚豪1,周帅琪1,邵晨晨1   

  1. 中国中医科学院附属望京医院,1骨伤综合科,3脊柱二科,4学术发展处,北京市  100102;2中医正骨技术北京市重点实验室,北京市  100700
  • 收稿日期:2021-12-09 接受日期:2022-01-28 出版日期:2023-01-18 发布日期:2022-06-18
  • 通讯作者: 展嘉文,副主任医师,中国中医科学院附属望京医院骨伤综合科,北京市 100102;中医正骨技术北京市重点实验室,北京市 100102 朱立国,主任医师,中国中医科学院望京医院脊柱二科,北京市 100102;中医正骨技术北京市重点实验室,北京市 100102
  • 作者简介:张伟业,男,1992年生,河北省保定市人,汉族,中国中医科学院在读博士,主要从事脊柱与相关疾病研究。
  • 基金资助:
    国家自然科学基金重点项目(81930118),项目负责人:朱立国;国家自然科学基金面上项目(81774330),项目负责人:冯敏山;国家自然科学基金青年项目(81804120),项目负责人:展嘉文;国家中医药管理局中医药创新团队及人才支持计划项目(ZYYCXTD-C-202003),项目负责人:朱立国;中国中医科学院基本科研业务费优秀青年科技人才(创新类)培养专项(ZZ13-YQ-038),项目负责人:展嘉文

Effect of nucleus pulposus cells-derived exosomes under cyclic mechanical tension on endplate chondrocytes

Zhang Weiye1, Zhan Jiawen1, 2, Zhu Liguo2, 3, Wang Shangquan1, 2, Chen Ming1, 2, Wei Xu2, 4, Feng Minshan3, 4, Yu Jie3, 4, Han Tao1, 4, Cai Chuhao1, Zhou Shuaiqi1, Shao Chenchen1   

  1. 1Department of Orthopedics and Traumatology, 3Second Department of Spine, 4Academic Development Department, Wangjing Hospital, Chinese Academy of Traditional Chinese Medicine, Beijing 100102, China; 2Beijing Key Laboratory of Bone Setting Technology of Traditional Chinese Medicine, Beijing 100700, China
  • Received:2021-12-09 Accepted:2022-01-28 Online:2023-01-18 Published:2022-06-18
  • Contact: Zhan Jiawen, Associate chief physician, Department of Orthopedics and Traumatology, Wangjing Hospital, Chinese Academy of Traditional Chinese Medicine, Beijing 100102, China; Beijing Key Laboratory of Bone Setting Technology of Traditional Chinese Medicine, Beijing 100700, China Zhu Liguo, Chief physician, Beijing Key Laboratory of Bone Setting Technology of Traditional Chinese Medicine, Beijing 100700, China; Second Department of Spine, Wangjing Hospital, Chinese Academy of Traditional Chinese Medicine, Beijing 100102, China
  • About author:Zhang Weiye, MD candidate, Department of Orthopedics and Traumatology, Wangjing Hospital, Chinese Academy of Traditional Chinese Medicine, Beijing 100102, China
  • Supported by:
    the National Natural Science Foundation of China, Nos. 81930118 (to ZLG), 81774330 (to FMS), and 81804120 (to ZJW); Innovation Team and Talents Cultivation Program of the National Administration of Traditional Chinese Medicine, No. ZYYCXTD-C-202003 (to ZLG); the Fundamental Research Funds for Young Scientific Talents in Chinese Academy of Traditional Chinese Medicine, No. ZZ13-YQ-038 (to ZJW)

摘要:

文题释义:
外泌体:特指包含了复杂RNA和蛋白质的直径在40-100 nm的盘状囊泡,天然存在于体液中,包括血液、唾液、尿液、脑脊液和乳汁等,其功能取决于其所来源的细胞类型,可参与到机体免疫应答、抗原提呈、细胞迁移、细胞分化、肿瘤侵袭等方面。有关椎间盘不同组织细胞外泌体的分泌、摄取及其内容物、“运载物”和相应的功能尚需深入研究。
软骨终板:是椎间盘组成的重要部分。椎体终板是椎体在生长发育过程中椎体上下面的骨骺板骨化停止后形成的骨板,呈轻度凹陷,即为骨性终板。椎体终板的中央仍为一薄层透明软骨覆盖,并终生存在,即为软骨终板,上下软骨终板与髓核和纤维环连接共同构成椎间盘,软骨终板的完整性与功能对维持椎间盘生理作用有重要意义。

背景:终板软骨细胞在髓核-终板复杂的微环境中受到多种因素的调节,异常应力所引发的微环境改变对终板软骨细胞的影响尚需深入探究。
目的:观察循环牵张力条件下髓核细胞外泌体的分泌情况,以及应力刺激髓核外泌体对终板软骨细胞的影响。
方法:体外分离培养人腰椎髓核细胞,应用FX-5000T系统对髓核细胞进行循环牵张力加载,采用磁珠法提取应力与非应力条件下的髓核细胞外泌体,流式细胞术检测外泌体纯度与浓度。以PKH67荧光染料标记外泌体,将应力与非应力条件下的髓核细胞外泌体分别与终板软骨细胞共孵育24 h,以单独培养的终板软骨细胞为对照,观察终板软骨细胞对外泌体的摄取情况、终板软骨细胞的凋亡和活性以及终板软骨细胞基质中相关基因的mRNA表达。
结果与结论:①一定条件的循环牵张力刺激可促进髓核细胞外泌体分泌,且该条件下的外泌体颗粒浓度高于非应力条件;②倒置荧光显微镜下可见,应力与非应力条件下的髓核细胞外泌体均可被终板软骨细胞摄取;③CCK8与流式细胞术检测显示,应力组终板软骨细胞的活性低于对照组(P < 0.05)、凋亡率高于对照组(P < 0.05);④RT-PCR检测显示,与对照组比较,非应力组基质金属蛋白酶3、骨形态发生蛋白2 mRNA表达增加(P < 0.05);与对照组比较,应力组Ⅱ型胶原mRNA表达降低(P < 0.05),解聚蛋白样金属蛋白酶5、基质金属蛋白酶3及β-连环蛋白mRNA表达升高(P < 0.05);与非应力相比,应力组Ⅱ型胶原mRNA表达降低(P < 0.05),解聚蛋白样金属蛋白酶5、基质金属蛋白酶3及骨形态发生蛋白2 mRNA表达升高(P < 0.05);⑤结果表明一定条件的应力可促进髓核细胞外泌体分泌,且该条件下的外泌体可抑制终板软骨细胞的活性,造成细胞基质合成与分解代谢紊乱。

https://orcid.org/0000-0002-0583-0383 (张伟业)

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: 循环牵张力, 髓核细胞, 外泌体, 终板软骨细胞, 微环境, 椎间盘, 退行性变, 异常应力

Abstract: BACKGROUND: Endplate chondrocytes are regulated by many factors in the microenvironment of the intervertebral disc and the influence of microenvironmental changes inside the intervertebral disc caused by abnormal stress on endplate chondrocytes still needs to be explored.
OBJECTIVE: To observe the exosome secretion of nucleus pulposus cells under cyclic mechanical tension conditions and the effect of exosomes on endplate chondrocytes under this condition.
METHODS: Nucleus pulposus cells were isolated and cultured from the nucleus pulposus of patients undergoing lumbar surgery in vitro. The cells were loaded with cyclic mechanical tension by FX-5000T system. Exosomes secreted from nucleus pulposus cells under stress and non-stress conditions were extracted by magnetic bead method. Purity and concentration of nucleus pulposus cells-derived exosomes were identified by flow cytometry. The exosomes were labeled with PKH67 fluorescent dye and incubated with endplate chondrocytes for 24 hours. Endplate chondrocytes cultured alone were used as control. The uptake of exosomes by endplate chondrocytes was observed under inverted fluorescence microscope. Then the apoptosis and activity of endplate chondrocytes were observed. Type II collagen, depolymerized protein like metalloproteinase 5, matrix metalloproteinase 3, bone morphogenetic protein 2, and β-catenin mRNA expression were detected. 
RESULTS AND CONCLUSION: Under certain cyclic mechanical tension conditions, nucleus pulposus cells could be promoted to secrete exosomes, and the particle concentration of nucleus pulposus cells-derived exosomes was higher than that under non-stress conditions. Under the inverted fluorescence microscope, both stress and non-stress nucleus pulposus cells-derived exosomes could be ingested by endplate chondrocytes. Compared with the control group, the viability of endplate chondrocytes decreased significantly (P < 0.05) and the apoptosis rate increased in the stress group (P < 0.05). Compared with the control group, the mRNA expression of matrix metalloproteinase 3 and bone morphogenetic protein 2 in endplate chondrocytes decreased significantly in the non-stress group (P < 0.05). Compared with the control group, the mRNA expression of type I collagen decreased significantly in the stress group (P < 0.05), while the mRNA expression of depolymerized protein like metalloproteinase 5, matrix metalloproteinase 3 and β-catenin increased significantly in the stress group (P < 0.05). Compared with the non-stress group, the mRNA expression of type II collagen decreased significantly in the stress group, while the mRNA expression of depolymerized protein like metalloproteinase 5, matrix metalloproteinase 3 and bone morphogenetic protein 2 increased significantly in the stress group (P < 0.05). Therefore, abnormal stress could promote the exosome secretion of nucleus pulposus cells, and the exosomes under this condition could inhibit the viability of endplate chondrocytes and cause the disorder of cell matrix synthesis and catabolism.

Key words: cyclic mechanical tension, nucleus pulposus cell, exosome, endplate chondrocyte, microenvironment, intervertebral disc, degeneration, abnormal stress

中图分类号: