The experimental study of fluid flow shear stress promotes apoptosis of rat condylar chondrocytes

doi:10.3969/j.issn.1005-4979.2021.02.001

《Journal of Oral and Maxillofacial Surgery》 ›› 2021, Vol. 31 ›› Issue (2): 69-74. doi: 10.3969/j.issn.1005-4979.2021.02.001

• Basic Scientific Study • Next Articles

The experimental study of fluid flow shear stress promotes apoptosis of rat condylar chondrocytes

XIE Mianjiao¹(), ZHOU Peng³, DUAN Jing¹, MA Qin²(), WANG Meiqing¹()

¹ State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi International Joint Research Center for Oral Diseases, Department of Oral Anatomy and Physiology, The Third Hospital, Air Force Medical University, Xi’an 710032, Shaanxi Province
² State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi International Joint Research Center for Oral Diseases, Department of Oral Anatomy and Physiology, The Third Hospital, Department of Maxillofacial Surgery, Xi’an 710032, Shaanxi Province
³ Department of Basic Stomatology, School of Stomatology, Jiamusi University, Jiamusi 154007, Heilongjiang Province, China

Received:2020-11-22 Revised:2020-12-04 Online:2021-04-28 Published:2021-04-26

流体剪切力促进大鼠髁突软骨细胞凋亡的实验研究

谢勉姣¹(), 周鹏³, 段晶¹, 马秦²(), 王美青¹()

¹ 军事口腔医学国家重点实验室，国家口腔疾病临床医学研究中心，陕西省口腔疾病国际联合研究中心，空军军医大学第三附属医院口腔解剖生理学教研室，陕西西安 710032
² 军事口腔医学国家重点实验室，国家口腔疾病临床医学研究中心，陕西省口腔疾病国际联合研究中心，空军军医大学第三附属医院颌面外科，陕西西安 710032
³ 佳木斯大学口腔医学院口腔基础医学教研室，黑龙江佳木斯 154007

通讯作者: 王美青，教授. E-mail: mqwang@fmmu.edu.cn；马秦，教授. E-mail: qma@fmmu.edu.cn
作者简介:
谢勉姣（1992—），女，陕西人，硕士研究生. E-mail: ncuskxiemianjiao@163.com
基金资助:
国家自然科学基金(81920108013)

Abstract

Abstract:

Objective: To investigate the apoptosis of rat primary mandibular condylar chondrocytes stimulated by the fluid flow shear stress(FFSS). Methods: The fluid flow shear stress of 16 dyn/cm² was delivered to the primary mandibular condylar chondrocytes for 0.5 h, 1 h, 2 h and 4 h respectively. The changes of intracellular Calcium ion(Ca²⁺) was tested by using the Calcium colorimetric assay kit, and the activity of Calpain was detected by using the Calpain activity fluorometric assay kit. The cytoskeleton was observed by fluorescence staining. The expression levels of apoptosis-related molecules such as Talin 1, Integrin β1, Bim, B-cell lymphoma-2(Bcl-2), Cleaved-Caspase-3 were measured by Western blot assay. Results: After stimulating primary mandibular condylar chondrocytes for different duration, the concentration of intracellular Ca²⁺ was increased and reached to the peak at 1 h, and persisted at high level till 4 h. The activity of Calpain was increasing and reached the peak at 1 hour, but decreased to the 0 h level at 4 h. The expression levels of F-actin and its related protein Talin and Integrin β1 were decreased. The expression level of apoptosis-related protein Bim was increased while Bcl-2 showed decrease. The apoptosis executive molecule Cleaved-Caspase-3 was gradually increased (all above P<0.05). Conclusion: The stimulation of FFSS induces Ca²⁺ influx of chondrocytes, stimulates Calpain activity, and enhances hydrolyzation of Talin protein, ultimately leads to apoptosis of chondrocytes.

Key words: chondrocytes, fluid flow shear stress, Calpain, Talin, apoptosis

摘要：

目的：观察流体剪切力（fluid flow shear stress，FFSS）刺激下大鼠原代髁突软骨细胞发生凋亡的特点。方法：采用16 dyn/cm²的FFSS刺激原代髁突软骨细胞0.5、1、2及4 h，用钙显色试剂盒检测细胞内钙离子（Ca²⁺）的变化；钙蛋白酶（Calpain）活性检测试剂盒检测Calpain的活性变化；荧光染色观察细胞骨架的变化；蛋白质印迹法（Western blot）检测踝蛋白1（Talin 1）、整合素β1（Integrin β1）、促凋亡调节蛋白Bim、B细胞淋巴瘤-2蛋白（B-cell lymphoma-2, Bcl-2）、活化形式的胱天蛋白酶-3（Cleaved-Caspase-3）等凋亡相关分子的表达水平变化。结果：FFSS刺激原代软骨细胞不同时长后，细胞内Ca²⁺浓度均显著上升，在1 h时达到峰值，并一直维持在较高的水平；Calpain的活性早期明显升高，于1 h达到峰值，但4 h时又降至0 h组水平；肌动蛋白（F-actin）及其相关的Talin和Integrin β1蛋白表达水平随加力时间的延长而降低；凋亡相关的Bim和Bcl-2蛋白表达水平分别呈现升高和降低的趋势；凋亡执行分子Cleaved-Caspase-3表达水平也逐渐升高（以上均P<0.05）。结论：FFSS刺激可升高细胞内Ca²⁺水平，激活Calpain，增加Talin蛋白的水解，最终导致软骨细胞凋亡。

关键词: 软骨细胞, 流体剪切力, 钙蛋白酶, 踝蛋白, 细胞凋亡

CLC Number:

R782

XIE Mianjiao, ZHOU Peng, DUAN Jing, MA Qin, WANG Meiqing. The experimental study of fluid flow shear stress promotes apoptosis of rat condylar chondrocytes[J]. 《Journal of Oral and Maxillofacial Surgery》, 2021, 31(2): 69-74.

谢勉姣, 周鹏, 段晶, 马秦, 王美青. 流体剪切力促进大鼠髁突软骨细胞凋亡的实验研究[J]. 《口腔颌面外科杂志》, 2021, 31(2): 69-74.

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URL: https://journal06.magtech.org.cn/Jweb_joms/EN/10.3969/j.issn.1005-4979.2021.02.001

https://journal06.magtech.org.cn/Jweb_joms/EN/Y2021/V31/I2/69

Figures/Tables 4

Figure 1 Stimulated by 16 dyn/cm² fluid flow shear stress, the changes of calcium content at different time points of 1 μg total protein primary chondrocytes

Figure 2 Changes of calpain activity of 50 μg total protein primary chondrocytes stimulated by 16 dyn/cm² fluid flow shear stress at different time points

Figure 3 The changes of cytoskeleton protein F-actin stimulated by 16 dyn/cm² fluid flow shear stress at different time points

Figure 4 Comparison of the expression level and its gray-scale value of Talin 1，Integrin β1, Bcl-2, Bim and Cleaved-Caspase-3 proteins stimulated by 16 dyn/cm² fluid flow shear stress at different time points observed by Western blot

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