Nd：YAP激光生物刺激通过WNT/β-catenin信号通路促进人牙周韧带细胞增殖和成骨分化

doi:10.3969/j.issn.1674-8115.2025.05.004

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (5): 562-569.doi: 10.3969/j.issn.1674-8115.2025.05.004

Nd：YAP激光生物刺激通过WNT/β-catenin信号通路促进人牙周韧带细胞增殖和成骨分化

许沐馨, 刘贤, 蒋立姗, 孙青()

江苏省常州市中医医院钟楼院区，江苏省常州市口腔医院牙周科，常州 213003

收稿日期:2024-10-17 接受日期:2024-11-25 出版日期:2025-05-28 发布日期:2025-05-28
通讯作者: 孙　青，副主任医师，硕士；电子信箱：jinghan6@163.com。
作者简介:许沐馨（1991—），女，主治医师，硕士；电子信箱：535192212@qq.com。
基金资助:
常州市卫生健康委员会重大科技项目(ZD202221)

Promotion of Nd:YAP laser biostimulation on the proliferation and osteogenic differentiation of human periodontal ligament cells through WNT/β-catenin signaling pathway

XU Muxin, LIU Xian, JIANG Lishan, SUN Qing()

Department of Periodontics, Zhonglou Branch of Changzhou Traditional Chinese Medicine Hospital/Changzhou Stomatological Hospital, Jiangsu Province, Changzhou 213003, China

Received:2024-10-17 Accepted:2024-11-25 Online:2025-05-28 Published:2025-05-28
Contact: SUN Qing, E-mail: jinghan6@163.com.
Supported by:
Major Science and Technology Project of Changzhou Health Commission(ZD202221)

摘要/Abstract

摘要：

目的·探讨掺钕钇铝钙钛矿（neodymium-doped yttrium aluminum perovskite，Nd：YAP）激光生物刺激对人牙周韧带细胞（human periodontal ligament cells，hPDLCs）增殖和成骨分化的影响及可能的机制。方法·于常州市口腔医院收集因正畸拔除的前磨牙5颗，取牙根中1/3处的牙周韧带组织体外培养hPDLCs。使用Nd：YAP激光治疗仪生物刺激功能［G（－）模式］照射细胞，根据照射时间将细胞分为对照组（无激光照射），以及5 s、10 s、15 s、20 s和30 s组。采用细胞计数试剂盒-8（cell counting kit-8，CCK-8）法检测各组hPDLCs增殖的情况；诱导成骨分化后，用碱性磷酸酶（alkaline phosphatase，ALP）染色试剂盒和活性检测试剂盒检测细胞ALP含量和活性水平，通过茜素红S染色和钙定量分析评价细胞的钙盐水平；实时荧光定量PCR（quantitative real-time PCR，qPCR）和Western blotting分析各组细胞WNT/β-连环蛋白（β-catenin）信号通路中dickkopf相关蛋白1（dickkopf-related protein 1，DKK-1）、β-catenin、runt相关转录因子2（runt-related transcription factor 2，RUNX2）基因和蛋白的表达。结果·CCK-8检测结果发现，从照射后3 d开始，10 s、15 s、20 s、30 s组细胞的增殖水平增强（均P<0.001）。诱导成骨分化后，激光照射组ALP含量及活性、钙盐水平都随照射时间延长，逐渐升高（均P<0.05）。qPCR和Western blotting分析结果显示：激光照射组DKK-1基因和蛋白表达水平随照射时间延长而降低；β-catenin和RUNX2基因和蛋白表达水平在照射时间延长后显著升高，15 s、20 s、30 s组与对照组差异均有统计学意义（均P<0.05）。结论·Nd：YAP激光生物刺激可能通过WNT/β-catenin信号通路促进hPDLCs增殖和成骨分化。

关键词: 掺钕钇铝钙钛矿激光, 激光生物刺激效应, 人牙周韧带细胞, 细胞增殖, 成骨分化

Abstract:

Objective ·To study the effect of neodymium-doped yttrium aluminum perovskite (Nd:YAP) laser biostimulation on the proliferation and osteogenic differentiation of human periodontal ligament cells (hPDLCs) and its possible mechanism. Methods ·Five premolars removed for orthodontic reasons were collected from Changzhou Stomatological Hospital, and the periodontal ligament tissues from the middle 1/3 of the roots were taken to culture hPDLCs in vitro. The cells were irradiated with the biostimulation function [G (－) mode] of the Nd:YAP laser. According to the irradiation time, the cells were divided into a control group (without laser irradiation), and groups irradiated for 5 s, 10 s, 15 s, 20 s and 30 s. The cell counting kit-8 (CCK-8) method was used to detect the proliferation of hPDLCs in each group. After osteogenic differentiation was induced, the alkaline phosphatase (ALP) content and activity level of the cells were detected using an ALP staining kit and an ALP activity detection kit. The calcium salt level of the cells was evaluated by alizarin red S staining and calcium quantitative analysis. The expression of genes and proteins related to the WNT/β-catenin signaling pathway, including dickkopf-related protein 1 (DKK-1), β-catenin, and runt-related transcription factor 2 (RUNX2), was analyzed by quantitative real-time PCR (qPCR) and Western blotting. Results ·The results of CCK-8 showed that the proliferation level of cells in the 10 s, 15 s, 20 s, and 30 s groups was enhanced from 3 d after irradiation (all P<0.05). After induction of osteogenic differentiation, ALP content, activity, and calcium salt level in the laser irradiation groups increased with the extension of irradiation time (all P<0.05). The results of qPCR and Western blotting analysis showed that the expression levels of the DKK-1 gene and protein in the laser irradiation groups decreased with the extension of irradiation time. However, the expression levels of β-catenin and RUNX2 genes and proteins increased significantly with the extension of irradiation time; there were statistically significant differences between the 15 s, 20 s, and 30 s groups and the control group (all P<0.05). Conclusion ·Nd:YAP laser biostimulation may promote proliferation and osteogenic differentiation of hPDLCs through WNT/β-catenin signaling pathway.

Key words: neodymium-doped yttrium aluminum perovskite laser (Nd:YAP laser), laser biostimulation effect, human periodontal ligament cell, cell proliferation, osteogenic differentiation

中图分类号:

R781.4

许沐馨, 刘贤, 蒋立姗, 孙青. Nd：YAP激光生物刺激通过WNT/β-catenin信号通路促进人牙周韧带细胞增殖和成骨分化[J]. 上海交通大学学报（医学版）, 2025, 45(5): 562-569.

XU Muxin, LIU Xian, JIANG Lishan, SUN Qing. Promotion of Nd:YAP laser biostimulation on the proliferation and osteogenic differentiation of human periodontal ligament cells through WNT/β-catenin signaling pathway[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(5): 562-569.

图/表 6

表1 用于qPCR的引物序列

Tab 1 Primer sequences used for qPCR

Gene	Forward primer (5′→3′)	Reverse primer (5′→3′)
DKK-1	CCTTGAACTCGGTTCTCAATTCC	CAATGGTCTGGTACTTATTCCCG
β-catenin	AAAATGGCAGTGCGTTTAG	TTTGAAGGCAGTCTGTCGTA
RUNX2	GTCTCACTGCCTCTCACTTG	CACACATCTCCTCCCTTCTG
β-actin	CATGTACGTTGCTATCCAGGC	CTCCTTAATGTCACGCACGAT

图1 Nd:YAP激光生物刺激对hPDLCs增殖水平的影响Note： Compared with the control group, the proliferation levels of the 10 s group (P<0.001), 15 s group (P<0.001), 20 s group (P<0.001), and 30 s group (P<0.001) were significantly different (n=3).

Fig 1 Effect of Nd:YAP laser biostimulation on proliferation of hPDLCs

图2 Nd:YAP激光生物刺激对成骨分化早期hPDLCs的ALP含量和活性的影响Note： A. ALP staining after osteogenic differentiation for 7 d (×40). B. ALP activity detection after osteogenic differentiation for 7 d (n=3). ①P=0.002, ②P<0.001, compared with the control group; ③P<0.001, compared with the 20 s group.

Fig 2 Effect of Nd:YAP laser biostimulation on ALP content and activity of hPDLCs during early osteogenic differentiation

图3 Nd:YAP激光生物刺激对成骨分化hPDLCs钙盐水平的影响Note： A. ARS staining after osteogenic differentiation for 21 d (×40). B. Quantitative determination of calcium levels after osteogenic differentiation for 21 d (n=3). ①P<0.001, compared with the control group; ②P=0.028, compared with the 20 s group.

Fig 3 Effect of Nd:YAP laser biostimulation on calcium level of hPDLCs during osteogenic differentiation

图4 Nd:YAP激光生物刺激对hPDLCs中 DKK-1、β-catenin和 RUNX2 基因表达的影响Note： ①P<0.001, compared with the control group; ②P=0.026, ③P=0.014, ④P<0.001, compared with the 20 s group. (n=3)

Fig 4 Effect of Nd:YAP laser biostimulation on expression of DKK-1,β-catenin and RUNX2 genes in hPDLCs

图5 Nd:YAP激光生物刺激对hPDLCs中DKK-1、β-catenin和RUNX2蛋白表达的影响Note： A. Protein bands in Western blotting. B. Quantitative analysis. ①P<0.001, compared with the control group; ②P<0.001, compared with the 15 s group; ③P<0.001, compared with the 10 s group; ④P<0.001, compared with the 20 s group. (n=3)

Fig 5 Effect of Nd:YAP laser biostimulation on expression of DKK-1, β-catenin and RUNX2 proteins in hPDLCs

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Nd：YAP激光生物刺激通过WNT/β-catenin信号通路促进人牙周韧带细胞增殖和成骨分化

Promotion of Nd:YAP laser biostimulation on the proliferation and osteogenic differentiation of human periodontal ligament cells through WNT/β-catenin signaling pathway

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