摘要
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。
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,andβ-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 bon
作者
张伟业
展嘉文
朱立国
王尚全
陈明
魏戌
冯敏山
于杰
韩涛
蔡楚豪
周帅琪
邵晨晨
Zhang Weiye;Zhan Jiawen;Zhu Liguo;Wang Shangquan;Chen Ming;Wei Xu;Feng Minshan;Yu Jie;Han Tao;Cai Chuhao;Zhou Shuaiqi;Shao Chenchen(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;Second Department of Spine,Wangjing Hospital,Chinese Academy of Traditional Chinese Medicine,Beijing 100102,China;Academic Development Department,Wangjing Hospital,Chinese Academy of Traditional Chinese Medicine,Beijing 100102,China)
出处
《中国组织工程研究》
CAS
北大核心
2023年第2期223-229,共7页
Chinese Journal of Tissue Engineering Research
基金
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。
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
cyclic mechanical tension
nucleus pulposus cell
exosome
endplate chondrocyte
microenvironment
intervertebral disc
degeneration
abnormal stress
