口腔医学研究

口腔医学研究 ›› 2021, Vol. 37 ›› Issue (5): 448-452.DOI: 10.13701/j.cnki.kqyxyj.2021.05.014

• 颞下颌关节病学研究 • 上一篇    下一篇

DDIT3调控TNF-α刺激下ATDC5细胞软骨向分化

杨畅, 许筱霄, 董晓菲, 董伟, 王家伟*   

  1. 口腔基础医学省部共建国家重点实验室培育基地和口腔生物医学教育部重点实验室,武汉大学口腔医学院 湖北 武汉 430079
  • 收稿日期:2021-01-08 发布日期:2021-05-17
  • 通讯作者: * 王家伟,E-mail:wb000238@whu.edu.cn
  • 作者简介:杨畅(1995~ ),女,浙江人,硕士在读,主要从事软骨内成骨相关的分子机制研究。
  • 基金资助:
    国家自然科学基金(编号:81870744)

DDIT3 Regulates Chondrogenic Differentiation of ATDC5 Cells Under TNF-α Stimulation

YANG Chang, XU Xiaoxiao, DONG Xiaofei, DONG Wei, WANG Jiawei*   

  1. The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan 430079, China
  • Received:2021-01-08 Published:2021-05-17

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摘要： 目的:探究在肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)刺激下,DNA损伤诱导转录因子3(DNA damage inducible transcript 3,DDIT3)对ATDC5细胞软骨向分化的调控。方法:不同浓度TNF-α (0、1、10、20 ng/mL)刺激下诱导ATDC5细胞分化7 d,同时在10 ng/mL TNF-α刺激下诱导慢病毒稳定转染ATDC5细胞分化7 d,qRT-PCR检测炎症相关因子环氧化酶(cyclooxygenase,COX)2、白细胞介素(interleukin,IL)-6、一氧化氮合酶(nitric oxide synthase,NOS)2、基质金属蛋白酶(matrix metalloproteinase,MMP)-13的表达,Western blot检测DDIT3、软骨向分化指标Sox9、Col10a1以及p-AKT、AKT蛋白表达情况,阿尔新蓝染色检测细胞外基质形成情况。结果:不同浓度TNF-α刺激下诱导ATDC5细胞分化引起COX2、IL-6、NOS2、MMP-13和DDIT3表达明显升高,Sox9、X型胶原蛋白(collagen type X alpha 1 chain,Col10a1)的表达下降。敲低DDIT3部分逆转了10 ng/mL TNF-α刺激导致的Sox9、Col10a1的表达下降和细胞外基质形成减少,同时促进磷酸化蛋白激酶B(phosphorylated protein kinase B,AKT)蛋白表达。结论:在TNF-α诱导的炎症微环境中,DDIT3调控了ATDC5细胞软骨向分化。

关键词: DNA损伤诱导转录因子3, 肿瘤坏死因子-α, ATDC5细胞, 软骨向分化

Abstract: Objective: To investigate the function of DDIT3 in the regulation of chondrogenic differentiation of ATDC5 cells under TNF-α stimulation. Methods: Wild-type ATDC5 cells were induced to chondrogenic differentiation for 7 days under the stimulation of various concentrations of TNF-α (0, 1, 10, 20 ng/mL), and then lentivirus-transfected ATDC5 cells were treated with chondrogenic induction medium contained 10ng/mL TNF-α for 7 days. qRT-PCR was used to detect mRNA changes of inflammation-related genes COX2, IL-6, NOS2, and MMP-13. Western blot was utilized to evaluate the protein expression levels of DDIT3 and chondrogenic related markers Sox9, Col10a1 p-AKT, and AKT. Alcian blue staining was conducted to detect extracellular matrix. Results: The expression of DDIT3 and inflammation-related factors COX2, IL-6, NOS2, and MMP-13 were increased in ATDC5 cells under chondrogenic induction in various concentrations of TNF-α after 7 days, while the chondrogenic differentiation markers Sox9 and Col10a1 were decreased. Knockdown of DDIT3 partly reversed the decreased expression levels of Sox9 and Col10a1 and extracellular matrix formation in ATDC5 cells under TNF-α stimulation, while promoted the expression level of p-AKT. Conclusion: Under TNF-α induced inflammatory microenvironment, DDIT3 could regulate chondrogenic differentiation of ATDC5 cells

Key words: DDIT3, TNF-α, ATDC5 cells, chondrogenic differentiation