流体剪切力促进大鼠髁突软骨细胞凋亡的实验研究

doi:10.3969/j.issn.1005-4979.2021.02.001

《口腔颌面外科杂志》 ›› 2021, Vol. 31 ›› Issue (2): 69-74. doi: 10.3969/j.issn.1005-4979.2021.02.001

• 基础研究 • 下一篇

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

收稿日期:2020-11-22 修回日期:2020-12-04 出版日期:2021-04-28 发布日期:2021-04-26
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)

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

摘要/Abstract

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

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

中图分类号:

R782

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.

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.

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

图/表 4

图1 原代软骨细胞在16 dyn/cm²的FFSS刺激下，总蛋白含量为1 μg的细胞中钙离子含量在不同加力时长下的变化情况 ***表示P<0.001，与对照组比较；****表示P<0.000 1，与对照组比较。

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

图2 不同时长16 dyn/cm²的FFSS刺激下，每50 μg总蛋白含量的原代软骨细胞内Calpain的活性变化 **表示P<0.01，与对照组比较； ****表示P<0.000 1，与对照组比较。

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

图3 不同时长16 dyn/cm²的流体剪切力刺激下，细胞骨架F-actin的变化情况 A. 原代软骨细胞在16 dyn/cm²的流体剪切力刺激0（对照组）、0.5、1、2和4 h时，F-actin的染色结果（×400）；B. 各时间点时F-actin平均荧光强度比较。***表示P<0.001，与对照组比较； ****表示P<0.000 1，与对照组比较。

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

图4 Western blot检测不同时长16 dyn/cm²的流体剪切力刺激下，Talin 1、Integrin β1、Bcl-2、Bim以及Cleaved-Caspase3的蛋白水平表达情况及其灰度值组间比较 A. 16 dyn/cm²的FFSS作用于原代软骨细胞0（对照组）、0.5、1、2和4 h，Talin 1、Integrin β1、Bcl-2、Bim以及Cleaved-Caspase-3蛋白表达水平的检测结果；B~F. 这些分子的统计分析图。**表示P<0.01，与对照组比较；***表示P<0.001，与对照组比较；****表示P<0.000 1，与对照组比较。

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

参考文献 20

[1]	Huang WJ, Warner M, Sasaki H, et al. Layer dependence in strain distribution and chondrocyte damage in porcine articular cartilage exposed to excessive compressive stress loading[J]. J Mech Behav Biomed Mater, 2020, 112: 104088. doi: 10.1016/j.jmbbm.2020.104088 URL
[2]	Zhang M, Yang H, Lu L, et al. Matrix replenishing by BMSCs is beneficial for osteoarthritic temporomandibular joint cartilage[J]. Osteoarthr Cartil, 2017, 25(9): 1551-1562. doi: 10.1016/j.joca.2017.05.007 URL
[3]	Ren HT, Yang HX, Xie MJ, et al. Chondrocyte apoptosis in rat mandibular condyles induced by dental occlusion due to mitochondrial damage caused by nitric oxide[J]. Arch Oral Biol, 2019, 101: 108-121. doi: S0003-9969(18)30844-6 pmid: 30927660
[4]	Yang H, Wen Y, Zhang M, et al. MTORC1 coordinates the autophagy and apoptosis signaling in articular chondrocytes in osteoarthritic temporomandibular joint[J]. Autophagy, 2020, 16(2): 271-288. doi: 10.1080/15548627.2019.1606647 pmid: 31007149
[5]	Loeser RF. Integrins and chondrocyte-matrix interactions in articular cartilage[J]. Matrix Biol, 2014, 39: 11-16. doi: 10.1016/j.matbio.2014.08.007 pmid: 25169886
[6]	Critchley DR. Cytoskeletal proteins talin and vinculin in integrin-mediated adhesion[J]. Biochem Soc Trans, 2004, 32(Pt 5): 831-836. doi: 10.1042/BST0320831 URL
[7]	Kim YN, Koo KH, Sung JY, et al. Anoikis resistance: An essential prerequisite for tumor metastasis[J]. Int J Cell Biol, 2012: 306879.
[8]	Goll DE, Thompson VF, Li H, et al. The calpain system[J]. Physiol Rev, 2003, 83(3): 731-801. doi: 10.1152/physrev.00029.2002 pmid: 12843408
[9]	Franco SJ, Rodgers MA, Perrin BJ, et al. Calpain-mediated proteolysis of talin regulates adhesion dynamics[J]. Nat Cell Biol, 2004, 6(10): 977-983. doi: 10.1038/ncb1175 pmid: 15448700
[10]	Kerstein PC, Jacques-Fricke BT, Rengifo J, et al. Mechanosensitive TRPC1 channels promote calpain proteolysis of talin to regulate spinal axon outgrowth[J]. J Neurosci, 2013, 33(1): 273-285. doi: 10.1523/JNEUROSCI.2142-12.2013 pmid: 23283340
[11]	Heinegard D, Oldberg A. Structure and biology of cartilage and bone matrix noncollagenous macromolecules[J]. FASEB J, 1989, 3(9): 2042-2051. pmid: 2663581
[12]	Ahrens PB, Solursh M, Reiter RS. Stage-related capacity for limb chondrogenesis in cell culture[J]. Dev Biol, 1977, 60(1): 69-82. pmid: 198274
[13]	Zhang J, Zhang HY, Zhang M, et al. Connexin43 hemichannels mediate small molecule exchange between chondrocytes and matrix in biomechanically-stimulated temporomandibular joint cartilage[J]. Osteoarthr Cartil, 2014, 22(6): 822-830. doi: 10.1016/j.joca.2014.03.017 URL
[14]	Nguyen C, Lieberherr M, Bordat C, et al. Intracellular calcium oscillations in articular chondrocytes induced by basic calcium phosphate crystals lead to cartilage degradation[J]. Osteoarthr Cartil, 2012, 20(11): 1399-1408. doi: 10.1016/j.joca.2012.07.017 URL
[15]	Storr SJ, Thompson N, Pu X, et al. Calpain in breast cancer: Role in disease progression and treatment response[J]. Pathobiology, 2015, 82(3-4): 133-141. doi: 10.1159/000430464 pmid: 26330354
[16]	Storr SJ, Carragher NO, Frame MC, et al. The calpain system and cancer[J]. Nat Rev Cancer, 2011, 11(5): 364-374. doi: 10.1038/nrc3050 pmid: 21508973
[17]	Liu X, Schnellmann RG. Calpain mediates progressive plasma membrane permeability and proteolysis of cytoskeleton-associated paxillin, talin, and vinculin during renal cell death[J]. J Pharmacol Exp Ther, 2003, 304(1): 63-70. pmid: 12490576
[18]	Fletcher DA, Mullins RD. Cell mechanics and the cytoskeleton[J]. Nature, 2010, 463(7280): 485-492. doi: 10.1038/nature08908
[19]	Parsons JT, Horwitz AR, Schwartz MA. Cell adhesion: Integrating cytoskeletal dynamics and cellular tension[J]. Nat Rev Mol Cell Biol, 2010, 11(9): 633-643. doi: 10.1038/nrm2957
[20]	Galluzzi L, Vitale I, Abrams JM, et al. Molecular definitions of cell death subroutines: Recommendations of the Nomenclature Committee on Cell Death 2012[J]. Cell Death Differ, 2012, 19(1): 107-120. doi: 10.1038/cdd.2011.96 pmid: 21760595

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