高浓度氟对人牙周膜干细胞凋亡的影响

doi:10.19439/j.sjos.2021.04.005

上海口腔医学 ›› 2021, Vol. 30 ›› Issue (4): 360-366.doi: 10.19439/j.sjos.2021.04.005

高浓度氟对人牙周膜干细胞凋亡的影响

汤颖¹, 沈忆芬¹, 刘超¹, 邱吟枫¹, 顾永春^1,*, 于金华^2,*

1.苏州市第九人民医院口腔科,江苏苏州 215200;
2.江苏省口腔医院牙体牙髓病科, 江苏南京 210029

收稿日期:2019-12-09 修回日期:2020-03-17 出版日期:2021-08-25 发布日期:2021-09-23
通讯作者: 顾永春,E-mail:guyc7152@163.com;于金华,E-mail:yujinhua@njmu.edu.cn。^*共同通信作者
作者简介:汤颖（1987-）,女,硕士,E-mail:1084616711@qq.com
基金资助:
江苏省口腔疾病研究重点实验室开放课题(JSK-LOD-KF-1705）; 苏州市吴江区卫健委科教兴卫项目（wwk201705、wwk202008）

Effect of high-concentration fluoride on apoptosis of human periodontal ligament stem cells

TANG Ying¹, SHEN Yi-fen¹, LIU Chao¹, QIU Yin-feng¹, GU Yong-chun¹, YU Jin-hua²

1. Ninth People's Hospital of Suzhou. Suzhou 215200;
2. Department of Endodontics, School of Stomatology, Nanjing Medical University. Nanjing 210029, Jiangsu Province, China

Received:2019-12-09 Revised:2020-03-17 Online:2021-08-25 Published:2021-09-23

摘要/Abstract

摘要： 目的: 探讨高浓度氟对人牙周膜干细胞（periodontal ligament stem cells, PDLSCs）凋亡的影响。方法: 从新鲜拔除的恒牙牙周膜中分离获得PDLSCs,分别用不同浓度的氟化钠（0~40 ppm F）处理细胞,CCK-8法检测细胞活力,Annexin V-PI染色及JC-1染色后,流式细胞仪分析、检测氟对PDLSCs细胞凋亡及线粒体膜电位的影响。H-E染色细胞爬片观察氟对细胞形态的影响,免疫荧光染色及共聚焦显微镜观察细胞色素C（cyt-c）,cleaved-caspase -9、-3的表达。RT-PCR检测caspase-9和-3的mRNA蛋白表达水平。Western印迹法分析丝裂原活化蛋白激酶家族（MAPKs）中ERK、JNK、p36总蛋白及磷酸化蛋白的表达水平。采用SPSS 13.0软件包对数据进行统计学分析。结果: 氟处理能抑制PDLSCs活力（CCK-8分析）,诱导细胞凋亡（Annexin V-PI染色）,其效应表现为浓度及时间依赖性。氟浓度≥10 ppm时,能明显诱导细胞线粒体膜电位去极化（JC-1染色）。免疫荧光分析显示,氟暴露促进cyt-c从线粒体释放到胞质,促进cleaved-caspase -9及-3 蛋白表达。RT-PCR同样证明,氟暴露上调caspase-9及-3的mRNA表达水平,呈浓度依赖性。Western印迹分析显示,氟能诱导MARK/ERK磷酸化激活,而JNK、p38磷酸化水平无显著变化,ERK特异性阻断剂U0126预处理能部分挽救PDLSCs的凋亡。结论: 高浓度氟通过内源性线粒体凋亡通路诱导PDLSCs细胞凋亡,MARK/ERK磷酸化部分参与其凋亡机制。

关键词: 牙周膜间充质干细胞, 氟, 细胞凋亡, 内源性凋亡通路

Abstract: PURPOSE: To explore the effects of high-concentration fluoride(F) on apoptosis of human periodontal ligament stem cells (PDLSCs). METHODS: PDLSCs were isolated from periodontal ligament tissues of extracted third molars, and treated with different concentrations (0-40 ppm F) of NaF for indicated period of time. CCK-8 assay was performed to detect cell viability. After stained with Annexin V-PI and JC-1, cell apoptosis and mitochondrial membrane potential were analyzed by flow cytometry. Immunofluorescence staining and confocal microscopic assay were used to detect the protein expression level of cyt-c, cleaved-caspase-9 and -3. The mRNA level of caspase -9 and -3 were examined by RT-PCR. The protein expression level of total and phosphate-ERK, JNK and p38 were analyzed by Western blot. SPSS 13.0 software package was used for statistical analysis. RESULTS: Fluoride treatment inhibited cell viability (CCK-8 assay) and induced apoptosis of PDLSCs (Annexin V-PI staining) in a dose- and time-dependent manner. Immunofluorescence assay showed that fluoride with a dose ≥10 ppm significantly induced release of cyt-c from the mitochondria to cytosol, and up-regulation of expression of cleaved-caspase -9 and -3. RT-PCR confirmed that the mRNA level of caspase-9 and -3 increased with the dose of fluoride. Western blot assay confirmed that fluoride induced up-regulation of p-ERK, but not that of p-JNK and p-p38, and specifically blocking ERK pathway with U0126 could partially rescue the fluoride-induced cell apoptosis. CONCLUSIONS: High concentrations of fluoride induces apoptosis of PDLSCs via intrinsic mitochondrial pathway, and phosphation of MAPK/ERK is involved in the F-induce cell apoptosis.

Key words: Periodontal ligament stem cells, Fluoride, Apoptosis, Intrinsic mitochondrial apoptotic pathway

中图分类号:

R781.4

汤颖, 沈忆芬, 刘超, 邱吟枫, 顾永春, 于金华. 高浓度氟对人牙周膜干细胞凋亡的影响[J]. 上海口腔医学, 2021, 30(4): 360-366.

TANG Ying, SHEN Yi-fen, LIU Chao, QIU Yin-feng, GU Yong-chun, YU Jin-hua. Effect of high-concentration fluoride on apoptosis of human periodontal ligament stem cells[J]. Shanghai Journal of Stomatology, 2021, 30(4): 360-366.

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高浓度氟对人牙周膜干细胞凋亡的影响

Effect of high-concentration fluoride on apoptosis of human periodontal ligament stem cells

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