自噬通过GATA4调控人牙髓细胞衰老反应

doi:10.13701/j.cnki.kqyxyj.2020.12.014

口腔医学研究 ›› 2020, Vol. 36 ›› Issue (12): 1142-1147.DOI: 10.13701/j.cnki.kqyxyj.2020.12.014

自噬通过GATA4调控人牙髓细胞衰老反应

黄佩, 乔玮玮, 孟柳燕^*

武汉大学口腔医学院口腔生物医学工程教育部重点实验室湖北武汉 430079

收稿日期:2020-05-18 出版日期:2020-12-28 发布日期:2020-12-28
通讯作者: *孟柳燕,E-mail:mengliuyan@whu.edu.cn
作者简介:黄佩(1995~ ),女,湖北随州人,硕士在读,主要从事牙髓炎症及修复方面研究。
基金资助:
国家自然科学基金(编号:81870761)

Autophagy Regulates Senescence in Human Dental Pulp Cells via GATA4

HUANG Pei, QIAO Weiwei, MENG Liuyan^*

Key Laboratory of Oral Biomedical Engineering of Ministry of Education,School of Stomatology,Wuhan University,Wuhan 430079,China

Received:2020-05-18 Online:2020-12-28 Published:2020-12-28

摘要/Abstract

摘要： 目的:探究脂多糖(lipopolyasccharide,LPS)诱导的炎症微环境下,自噬对人牙髓细胞(human dental pulp cells,hDPCs)衰老反应的调控作用。方法:体外分离培养hDPCs,10 μg/L LPS连续刺激hDPCs 6 d,并分别利用自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)与GATA结合蛋白4(GATA binding protein 4,GATA4)小干扰RNA(small interfering RNA,siRNA)进一步处理hDPCs。Western blot检测不同处理条件下hDPCs内GATA4、微管相关蛋白轻链3(microtubule-associated protein light chain 3,LC3)、衰老相关蛋白p53及p16的表达变化。细胞免疫荧光染色检测不同处理条件下LC3的表达,并对衰老相关β-半乳糖苷酶(senescence-associated β-galactosidase,SA-β-Gal)进行染色。结果:LPS连续刺激hDPCs后,LC3、GATA4、p53、p16的表达及SA-β-Gal阳性细胞数增加;3-MA抑制hDPCs自噬后,GATA4、p53及p16的表达及SA-β-Gal阳性细胞数进一步增加;敲低GATA4后,LC3表达无变化,而p53、p16的表达及SA-β-Gal阳性细胞数明显降低。结论:在LPS诱导的炎症微环境下,自噬通过GATA4负性调节hDPCs的衰老反应。

关键词: 人牙髓细胞, 炎症, 自噬, GATA结合蛋白4, 衰老

Abstract: Objective: To investigate how autophagy acts on senescence in human dental pulp cells (hDPCs) in lipopolysaccharide (LPS)-induced inflammatory microenvironment. Methods: hDPCs were isolated and cultured in vitro and continuously stimulated by 10 ng/L LPS for 6 days. Autophagy inhibitor 3-methyladenine (3-MA) and GATA binding protein 4 (GATA4) siRNA were respectively applied to treat hDPCs. Western blotting was used to detect the expression of GATA4, microtubule-associated protein light chain 3(LC3), and senescence-related proteins p53 and p16 in hDPCs under different treatments. Cell immunofluorescent assay was utilized to evaluate the expression of LC3. β-galactosidase staining kit was used to detect the expression of senescence-associated β-galactosidase (SA-β-Gal) in hDPCs. Results: After LPS stimulation, the expressions of LC3, GATA4, p53, and p16 as well as SA-β-Gal positive cells in hDPCs significantly increased compared with those in control group. After inhibition of autophagy by 3-MA, the expression of GATA4, p53, p16, and SA-β-Gal positive cells further increased. Compared to Nc group, GATA4 siRNA group experienced a dramatic decrease in p53 and p16 expression and SA-β-Gal positive cells, however, there was no significant change in LC3 expression. Conclusion: Under a LPS-induced inflammatory microenvironment, autophagy negatively regulates the senescence in hDPCs via GATA4.

Key words: hDPCs, inflammation, autophagy, GATA4, senescence

黄佩, 乔玮玮, 孟柳燕. 自噬通过GATA4调控人牙髓细胞衰老反应[J]. 口腔医学研究, 2020, 36(12): 1142-1147.

HUANG Pei, QIAO Weiwei, MENG Liuyan. Autophagy Regulates Senescence in Human Dental Pulp Cells via GATA4[J]. Journal of Oral Science Research, 2020, 36(12): 1142-1147.

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自噬通过GATA4调控人牙髓细胞衰老反应

Autophagy Regulates Senescence in Human Dental Pulp Cells via GATA4

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