膜电位对人牙囊干细胞成骨分化的作用及其电生理机制研究

doi:10.13701/j.cnki.kqyxyj.2025.05.010

口腔医学研究 ›› 2025, Vol. 41 ›› Issue (5): 413-419.DOI: 10.13701/j.cnki.kqyxyj.2025.05.010

膜电位对人牙囊干细胞成骨分化的作用及其电生理机制研究

顾煜婕^1,2, 杨宜丹^1,2, 廖思琪², 王荷一², 周蕊², 兰小蓉², 徐晓梅^1,2, 左东川³, 曾锦^1,2*

1.西南医科大学附属口腔医院正畸科四川泸州 646000;
2.西南医科大学口腔医学院,西南医科大学口颌面修复重建和再生实验室四川泸州 646000;
3.西南医科大学心血管医学研究所,医学电生理学教育部重点实验室四川泸州 646000

收稿日期:2024-11-19 出版日期:2025-05-28 发布日期:2025-05-26
通讯作者: ^*曾锦,E-mail:zengjin@swmu.edu.cn
作者简介:顾煜婕(1999~ ),女,四川泸州人,硕士,医师,研究方向:口腔颌面部骨缺损修复与再生。
基金资助:
国家自然科学基金(编号:81800303)四川省自然科学基金(编号:2023NSFSC1522,2022YFS0634)四川省医学会科研课题(编号:S22029)西南医科大学大学生创新创业训练计划项目(编号:S202310632175;2023460;2024365)

Effect of Membrane Potential on Osteogenic Differentiation of Human Dental Follicle Stem Cells and Its Electrophysiological Mechanisms

GU Yujie^1,2, YANG Yidan^1,2, LIAO Siqi², WANG Heyi², ZHOU Rui², LAN Xiaorong², XU Xiaomei^1,2, ZUO Dongchuan³, ZENG Jin^1,2*

1. Department of Orthodontics, The Affiliated Stomatological Hospital of Southwest Medical University, Luzhou 646000, China;
2. Oral and Facial Reconstruction and Regeneration Laboratory, Southwest Medical University, Luzhou 646000, China;
3. Institute of Cardiovascular Research, Key Laboratory of Medical Electrophysiology of Ministry of Education, Southwest Medical University, Luzhou 646000, China

Received:2024-11-19 Online:2025-05-28 Published:2025-05-26

摘要/Abstract

摘要： 目的: 探讨膜电位对人牙囊干细胞(human dental follicle stem cells,hDFCs)成骨分化的作用及其电生理机制。方法: 组织块结合酶消化法分离、培养hDFCs。构建载有人Kir2.1钾通道特异短发卡RNA(short hairpin RNA,shRNA)基因序列的慢病毒载体。采用慢病毒转染结合Kir2.1钾通道特异阻断剂(4-甲氧基苄基-1-萘基甲基-胺盐,ML133),通过成骨分化诱导、茜素红染色、定量逆转录聚合酶链反应(real-time reverse transcriptase-polymerase chain reaction,RT-qPCR)以及全细胞膜片钳实验方法,观察药物阻断Kir2.1钾通道的功能或抑制Kir2.1钾通道的表达对hDFCs成骨分化能力以及膜电位的影响。降低细胞外钾离子的浓度(1 mmol/L),通过钙离子成像观察促使膜电位超极化对细胞内钙离子浓度的影响。采用钙池操纵钙通道(store-operated Ca²⁺ channels,SOCs)阻断剂(La³⁺)以及内质网钙泵阻断剂毒胡萝卜素(Thapsigargin, TG),通过钙离子成像鉴定SOCs通道在hDFCs介导的钙离子内流,并评估其在膜电位超极化导致hDFCs胞内钙离子浓度变化中的作用。结果: 茜素红染色、RT-qPCR以及全细胞膜片钳结果显示,药物阻断Kir2.1钾通道的功能或抑制Kir2.1钾通道的表达能够抑制hDFCs成骨矿化能力、成骨相关基因(RUNT相关转录因子2、骨钙素)的表达,并能够逆转hDFCs成骨分化过程中发生的膜电位超极化。钙成像结果显示:(1)SOCs通道介导hDFCs的钙离子内流。(2)促使膜电位超极化能够引起hDFCs胞内钙离子浓度的升高,该作用可被药物阻断Kir2.1钾通道的功能或抑制Kir2.1钾通道的表达抑制;可通过移除细胞外钙离子抑制;可被SOCs通道阻断剂抑制。结论: Kir2.1钾通道介导的膜电位超极化参与hDFCs成骨分化的调控,其机制与hDFCs胞内钙离子浓度的升高有关,而SOCs通道介导的钙离子内流在这一过程中起到重要作用。

关键词: 人牙囊细胞, 成骨分化, Kir2.1通道, 膜电位超极化, 钙离子

Abstract: Objective: To explore the effect of membrane potential on osteogenic differentiation of human dental follicle stem cells (hDFCs) and its electrophysiological mechanisms. Methods: hDFCs were isolated and cultured from human dental follicle tissue by enzymatic digestion methods. To evaluate the effects of Kir2.1 channels on the osteogenic differentiation capacity and membrane potentials of hDFCs, osteogenic differentiation induction, Alizarin red staining, real-time reverse transcriptase-polymerase chain reaction (RT-qPCR), and whole cell patch clamp were conducted by using lentiviral particles carrying human Kir2.1 channels specific short hairpin RNA (shRNA) and Kir2.1 channels specific inhibitor (ML133). Ca²⁺ imaging was performed to identify the store-operated Ca²⁺ channels (SOCs) mediated Ca²⁺ influx in hDFCs by using La³⁺ (a SOCs inhibitor) and Thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), and to evaluate the effect of membrane potential hyperpolarization caused by decreased extracellular potassium level (1 mmol/L) on intracellular Ca²⁺ concentration. Results: Alizarin red staining, RT-qPCR, and patch clamp results showed that blocking the function of Kir2.1 channels or inhibition of Kir2.1 channels expression could inhibit the osteogenic differentiation ability of hDFCs and reverse the membrane potential hyperpolarization associated with osteogenic differentiation. Ca²⁺ imaging results showed that: (1) SOCs channels mediated Ca²⁺ influx in hDFCs. (2) Promotion of membrane hyperpolarization increased the intracellular Ca²⁺ levels in hDFCs, which could be inhibited by blocking the function of Kir2.1 channels or inhibition of Kir2.1 channels expression; by removal of extracellular Ca²⁺; or by blocking the function of SOCs channels. Conclusion: Kir2.1-linked membrane hyperpolarization controls osteogenic differentiation of hDFCs by increasing intracellular Ca²⁺ levels and SOCs channels-mediated Ca²⁺ influx plays an important role in this process.

Key words: human dental follicle cells, osteogenic differentiation, Kir2.1 channel, membrane hyperpolarization, calcium

顾煜婕, 杨宜丹, 廖思琪, 王荷一, 周蕊, 兰小蓉, 徐晓梅, 左东川, 曾锦. 膜电位对人牙囊干细胞成骨分化的作用及其电生理机制研究[J]. 口腔医学研究, 2025, 41(5): 413-419.

GU Yujie, YANG Yidan, LIAO Siqi, WANG Heyi, ZHOU Rui, LAN Xiaorong, XU Xiaomei, ZUO Dongchuan, ZENG Jin. Effect of Membrane Potential on Osteogenic Differentiation of Human Dental Follicle Stem Cells and Its Electrophysiological Mechanisms[J]. Journal of Oral Science Research, 2025, 41(5): 413-419.

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膜电位对人牙囊干细胞成骨分化的作用及其电生理机制研究

Effect of Membrane Potential on Osteogenic Differentiation of Human Dental Follicle Stem Cells and Its Electrophysiological Mechanisms

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