. 2022 Mar 25;40(2):139-147.

doi: 10.7518/hxkq.2022.02.003.

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

[Article in English, Chinese]

Peng Zhang^{1

2}, Dongchuan Zuo³, Siyu Mou², Yutong Zhong², Xiaoping Yuan^{1

2}, Jin Zeng^{1

2}

Affiliations

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.

PMID: 38597045
PMCID: PMC9002191
DOI: 10.7518/hxkq.2022.02.003

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

[Article in English, Chinese]

Peng Zhang et al. Hua Xi Kou Qiang Yi Xue Za Zhi. 2022.

. 2022 Mar 25;40(2):139-147.

doi: 10.7518/hxkq.2022.02.003.

Authors

Peng Zhang^{1

2}, Dongchuan Zuo³, Siyu Mou², Yutong Zhong², Xiaoping Yuan^{1

2}, Jin Zeng^{1

2}

Affiliations

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.

PMID: 38597045
PMCID: PMC9002191
DOI: 10.7518/hxkq.2022.02.003

Abstract
in English, Chinese

Objectives: 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.

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

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。.

Keywords: human dental follicle cell; inward rectifier potassium 2.1 channel; membrane potential hyperpolarization; osteogenic differentiation.

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Conflict of interest statement

利益冲突声明：作者声明本文无利益冲突。

Figures

**图 1. hDFC培养及鉴定 × 40**
Fig 1 Cultivation and identification of hDFCs × 40 A：hDFC的原代培养；B：纯化后的第三代细胞；C：矿化结节的茜素红染色。

**图 2. 细胞表面抗原表达的流式细胞术检测结果**
Fig 2 Results of cell surface antigen expression by flow cytometry A：CD73；B：CD90；C：CD105；D：CD31；E：CD45；F：CD3。

**图 3. Kir2.1基因KCNJ2的表达情况**
Fig 3 KCNJ2 expression of Kir2.1 channel A：RT-PCR；B：RT-qPCR，与对照组相比，**P<0.01。

**图 4. hDFC成骨诱导前后的膜电位变化**
Fig 4 Membrane potential changes of the hDFCs before and after osteogenic induction A：膜片钳记录的细胞膜电位；B：比较分析，a为诱导前，b为诱导后，***P<0.01。

**图 5. 茜素红染色结果 × 40**
Fig 5 Results of alizarin red staining × 40 A~D：OS、K⁺、CsCl、ML133组的培养皿；E~H：OS、K⁺、CsCl、ML133组的镜下图。

**图 6. 茜素红染色半定量分析结果**
Fig 6 Semi-quantitative analysis result of alizarin red staining 与OS组相比，**P<0.01。

**图 7. 碱性磷酸酶染色结果 × 40**
Fig 7 Results of alkaline phosphatase staining × 40 A~D：OS、K⁺、CsCl、ML133组的培养皿；E~H：OS、K⁺、CsCl、ML133组的镜下图。

**图 8. 碱性磷酸酶活性测定结果**
Fig 8 Activity measurement results of alkaline phosphatase 与OS组相比，**P<0.01。

**图 9. RT-qPCR检测成骨分化相关基因RUNX2、OCN的表达情况**
Fig 9 Detection of osteogenic differentiation-related genes RU-NX2 and OCN expression by RT-qPCR 与OS组相比，*P<0.05，**P<0.01。

**图 10. 钙离子荧光染色结果 × 40**
Fig 10 Results of calcium ion fluorescent staining × 40 A：细胞外钾离子的浓度为5.4 mmol·L⁻¹；B：细胞外钾离子的浓度为2 mmol·L⁻¹。

**图 11. F340/380比值的分析结果**
Fig 11 The analysis result of F340/380 ratio

See this image and copyright information in PMC

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

Affiliations

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

Authors

Affiliations

Abstract
in English, Chinese

Conflict of interest statement

Figures

References

Abstract in English, Chinese

Conflict of interest statement

Figures

References

Abstract
in English, Chinese