华西口腔医学杂志 ›› 2022, Vol. 40 ›› Issue (2): 139-147.doi: 10.7518/hxkq.2022.02.003

• 基础研究 • 上一篇    下一篇

内向整流钾2.1通道蛋白对人牙囊细胞成骨分化的影响及其机制研究

张鹏1,2(), 左东川3, 牟思宇2, 钟宇彤2, 袁小平1,2, 曾锦1,2()   

  1. 1.西南医科大学附属口腔医院正畸科,泸州 646000
    2.西南医科大学口颌面修复重建和再生实验室,泸州 646000
    3.西南医科大学电生理学教育部重点实验室,心血管医学研究所,泸州 646000
  • 收稿日期:2021-07-17 修回日期:2022-01-30 出版日期:2022-04-01 发布日期:2022-04-01
  • 通讯作者: 曾锦 E-mail:978137783@qq.com;zengjin@swmu.edu.cn
  • 作者简介:张鹏,硕士,E-mail:978137783@qq.com
  • 基金资助:
    西南医科大学校级项目(2021ZKMS012);泸州市-西南医科大学科技战略合作项目(2019LZXNYDJ01);国家自然科学基金(81800303);西南医科大学大学生创新创业训练计划项目(S202110632133)

Effect of inward rectifier potassium 2.1 channel on the osteogenic differentiation of human dental follicle cells and its mechanism

Zhang Peng1,2(), Zuo Dongchuan3, Mou Siyu2, Zhong Yutong2, Yuan Xiaoping1,2, Zeng Jin1,2()   

  1. 1.Dept. 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:2021-07-17 Revised:2022-01-30 Online:2022-04-01 Published:2022-04-01
  • Contact: Zeng Jin E-mail:978137783@qq.com;zengjin@swmu.edu.cn
  • Supported by:
    Southwest Medical University School Project(2021ZKMS012);Luzhou-Southwest Medical University Science and Technology Strategic Cooperation Project(2019LZXNYDJ01);The National Natural Science Foundation of China(81800303);Undergraduate Training Program for Innovation and Entrepreneurship(S202110632133);Corres-pondence: Zeng Jin, E-mail: zengjin@swmu.edu.cn

摘要: 目的

探讨内向整流钾(Kir)2.1通道蛋白对人牙囊细胞(hDFC)成骨分化的影响及其机制。

方法

组织块结合酶消化法分离、培养hDFC,流式细胞术鉴定细胞来源,成骨诱导鉴定hDFC成骨分化能力;逆转录聚合酶链反应(RT-PCR)检测Kir2.1编码基因KCNJ2在hDFC中的表达,定量逆转录聚合酶链反应(RT-qPCR)检测Kir2.1在hDFC成骨诱导前和诱导后KCNJ2基因表达量的变化;膜片钳技术检测hDFC在成骨诱导前和诱导后的细胞膜电位变化;提高细胞外钾离子的浓度(50 mmol·L-1)观察膜电位去极化对hDFC成骨分化能力的影响,应用Kir2.1钾通道阻断剂氯化铯(CsCl)和4-甲氧基苄基-1-萘基甲基-胺盐(ML133)观察阻断Kir2.1通道对hDFC成骨分化能力的影响,同时行RT-qPCR检测观察2种干预措施前后成骨分化相关基因[RUNX相关转录因子2(RUNX2)、骨钙素(OCN)]的表达变化情况。降低细胞外钾离子的浓度(2 mmol·L-1),钙离子成像检测膜电位超极化对细胞内钙离子浓度的变化。

结果

RT-PCR结果显示,hDFC表达Kir2.1通道基因KCNJ2;RT-qPCR结果显示,成骨诱导7 d后,KCNJ2在hDFC中的表达量上调;膜片钳结果显示,成骨诱导7 d后,hDFC膜电位由(-12±3.2)mV超极化为(-47±5.2)mV;茜素红和碱性磷酸酶染色结果显示,提高细胞外钾离子的浓度或阻断Kir2.1通道蛋白的功能,能够明显抑制hDFC的成骨矿化能力;钙离子成像结果显示,膜电位超极化能够引起细胞内钙离子浓度的增高。

结论

Kir2.1通道蛋白介导的膜电位超极化在hDFC成骨分化的过程中起重要的作用。

关键词: 人牙囊细胞, 内向整流钾2.1通道蛋白, 成骨分化, 膜电位超极化

Abstract: Objective

This study aims to explore the effect of inward rectifier potassium (Kir) 2.1 channel on the osteogenic differentiation of human dental follicle cells (hDFCs) and its mechanism.

Methods

hDFCs were isolated and cultured, and their source was verified by flow cytometry. Osteogenic differentiation ability of hDFCs was evaluated by osteogenic induction. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to detect the gene expression of Kir2.1 gene (KCNJ2) in hDFCs. Real-time quantitative PCR (RT-qPCR) was performed to detect the expression of the Kir2.1 gene (KCNJ2) in hDFCs before and after osteogenic induction. Patch clamp technique was conducted to record the membrane potential changes of hDFCs before and after osteogenic induction. Moreover, the effect on the osteogenic differentiation of hDFCs was confirmed by increasing the concentration of extracellular potassium ions (50 mmol·L-1). Kir2.1 channel blockers cesium chloride (CsCl) and C19H20CINO (ML133) were applied to determine the effect of the Kir2.1 potassium channel on the osteogenic differentiation of hDFCs. At the same time, RT-qPCR was used to observe the expression changes of osteogenic differentiation related genes Runx related transcription factor 2 (Runx2) and osteocalcin (OCN) before and after the two intervention measures. Calcium imaging was performed to observe the effect of membrane potential hyperpolarization caused by decreased extracellular potassium level (2 mmol·L-1) on intracellular calcium concentration.

Results

RT-PCR results showed that hDFCs expressed the Kir2.1 channel gene (KCNJ2). The RT-qPCR results showed that the KCNJ2 gene expression in hDFCs was upregulated 7 days after osteogenic induction. The patch clamp results showed that the membrane potential of hDFCs hyperpolarized to (-47±5.2) mV from (-12±3.2) mV. Alizarin red and alkaline phosphatase staining results showed that increasing the concentration of the extracellular potassium or blocking the function of the Kir2.1 channel significantly inhibited the osteogenic mineralization ability of hDFCs. The membrane potential hyperpolarization increased the intracellular calcium concentration in hDFCs.

Conclusion

Membrane potential hyperpolarization mediated by the Kir2.1 channel plays an important role in the osteogenic differentiation of hDFCs.

Key words: human dental follicle cell, inward rectifier potassium 2.1 channel, osteogenic differentiation, membrane potential hyperpolarization

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