滑膜细胞及软骨细胞释放HMGB1流体力学阈值的研究

Investigation of the hydromechanical threshold of high-mobility group box 1 release from synovial cells and chondrocytes

摘要

目的 探究流体流动剪切力（fluid flow shear stress，FFSS）下，滑膜细胞及软骨细胞释放高迁移率族蛋白1（high-mobility group box 1，HMGB1）时流体力学的阈值；阐明异常机械力刺激下，滑膜细胞及软骨细胞破坏的先后顺序，为了解颞下颌关节骨关节炎的发病机制及病理研究提供实验基础。方法 获得医院动物实验伦理委员会的审批，于SD大鼠膝关节获取滑膜组织及软骨组织块，消化培养获得滑膜细胞及软骨细胞，取3~4代滑膜细胞及软骨细胞，通过流体剪切力学装置对软骨及滑膜细胞施加FFSS，根据不同大小的FFSS值分组，分别采用1、3、5、10 dyn/cm2的FFSS刺激滑膜细胞1 h，采用4、8、12、16 dyn/cm2的FFSS刺激软骨细胞1 h，静息培养（0 dyn/cm2）作为对照组，观察细胞的形态学变化、免疫组化检测HMGB1及白细胞介素-1β（interleukin-1β，IL-1β）的细胞表达分布，ELISA检测上清液中HMGB1与IL-1β的水平；Western blot检测细胞HMGB1与IL-1β蛋白表达水平。结果 随着FFSS负载的增大，滑膜细胞及软骨细胞逐渐肿胀，破裂，且细胞数量减少。随着FFSS负载的增大，HMGB1与IL-1β的表达由胞核逐渐转移至胞浆。在滑膜细胞中，与对照组相比，1、3、5、10 dyn/cm2干预组中的HMGB1和IL-1β在上清液及细胞内的表达水平明显升高（P<0.01）。在软骨细胞中，与对照组相比，4、12、16 dyn/cm2 FFSS干预组中HMGB1在上清液中的表达水平增加（P<0.05），细胞HMGB1蛋白表达水平明显增加（P<0.01）；而在8 dyn/cm2 FFSS干预组中HMGB1在上清液中的表达水平明显增加（P<0.01），但细胞HMGB1蛋白表达减少（P<0.01）；与对照组相比，4、8、12、16 dyn/cm2 FFSS干预组中IL-1β在上清液中表达水平逐渐增加（P<0.01）；除4 dyn/cm2组外，随着FFSS负载增大，细胞中IL-1β的蛋白表达水平逐渐升高。结论 滑膜细胞及软骨细胞随FFSS负载增大出现溶胀破裂，释放HMGB1流体力学阈值分别接近于1 dyn/cm2和8 dyn/cm2；即在受到机械力刺激时，滑膜细胞先于软骨细胞损伤。

Abstract

Objective To investigate the effect of fluid flow shear stress (FFSS) on the fluid mechanic threshold of high-mobility group box 1 (HMGB1) release by synovial cells and chondrocytes. Moreover, the mechanism of chondrocyte and synovial cell damage induced by abnormal mechanical force was investigated to provide an experimental basis for exploring the pathogenesis and pathology of temporomandibular joint osteoarthritis. Methods With the approval of the Ethics Committee for Animal Experiments of the hospital, synovial tissue and cartilage tissue blocks were obtained from the knee joints of Sprague-Dawley (SD) rats, and synovial cells and chondrocytes were cultured and digested for subsequent experiments. Synovial cells and chondrocytes of 3-4 generations were acquired, and FFSS was applied to synovial and cartilage cells using a fluid shear mechanical device. The cells were divided according to the FFSS values of different sizes. Synovial cells were stimulated for 1 h with 1, 3, 5, or 10 dyn/cm² of FFSS, and chondrocytes were stimulated for 1 h with 4, 8, 12, or 16 dyn/cm² of FFSS. Resting cultures (0 dyn/cm²) were used as the control group. Changes in the morphology of the cells were observed. The expression and distribution of HMGB1 and interleukin-1β (IL-1β) were observed by immunohistochemistry. The expression of HMGB1 and IL-1β in the supernatant was analyzed by ELISA. The protein expression levels of intracellular HMGB1 and IL-1β were detected by Western blot. Results With increasing FFSS, the synovial cells and chondrocytes gradually swelled and ruptured, and the number of cells decreased. With increasing FFSS, the localizationof HMGB1 and IL-1β gradually shifted from the nucleus to the cytoplasm. In synovial cells, compared with those in the control group, the expression levels of HMGB1 and IL-1β were increased both in the supernatant and cells in the 1, 3, 5 and 10 dyn/cm² intervention groups (P<0.01). In chondrocytes, compared with those in the control group, the expression levels of HMGB1 in the supernatant were increased in the 4, 12 and 16 dyn/cm² intervention groups (P<0.05), and the protein expression levels of HMGB1 were significantly increased (P<0.01). The expression levels of HMGB1 in the supernatant were significantly increased in the 8 dyn/cm² intervention groups (P<0.01); however, the protein expression levels of HMGB1 were significantly decreased. Compared with those in the control group, the expression levels of IL-1β in the supernatant gradually increased in the 4, 8, 12 and 16 dyn/cm² intervention groups (P<0.01). With the exception of those in the 4 dyn/cm² group, the protein expression levels of IL-1β gradually increased with increasing FFSS (P<0.05). Conclusion With increasing FFSS, synovial cells and chondrocytes gradually swelled and burst, and the hydromechanical thresholds of HMGB1 release were 1 dyn/cm² and 8 dyn/cm², respectively. Therefore, upon stimulation with a mechanical force, synovial damage was damaged before chondrocytes.

关键词

颞下颌关节骨关节炎 / 滑膜细胞 / 软骨细胞 / 流体流动剪切力 / 阈值 / 高迁移率族蛋白1 / 白细胞介素-1β

Key words

temporomandibular joint osteoarthritis / synovial cells / chondrocytes / fluid flow shear stress / threshold / high-mobility group box 1 / interleukin-1β

Author summay

童乔莹，医师，硕士研究生，Email：[e-mail]