METTL3对炎症环境中根尖牙乳头干细胞增殖及迁移的调节作用

陈婷婷; 朱永娜; 吴越; 刘茜

doi:10.16766/j.cnki.issn.1674-4152.004082

METTL3对炎症环境中根尖牙乳头干细胞增殖及迁移的调节作用

doi: 10.16766/j.cnki.issn.1674-4152.004082

陈婷婷¹,
朱永娜¹,
吴越²,
刘茜^1, ,

1.
蚌埠医科大学第二附属医院口腔科, 安徽蚌埠 233040
2.
蚌埠医科大学口腔医学院, 安徽蚌埠 233030

基金项目:

安徽省高校自然科学研究项目 2023AH051997

详细信息

通讯作者:
刘茜，E-mail: 871190697@qq.com

中图分类号: R781.3 R-332
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出版历程
- 收稿日期: 2024-11-05
- 网络出版日期: 2025-10-25

The regulation of METTL3 on the proliferation and migration of SCAPs in the inflammatory environment

1.
Department of Stomatology, the Second Affiliated Hospital of Bengbu Medical University, Bengbu, Anhui 233040, China

摘要

摘要: 目的探索METTL3是否对炎症环境中根尖牙乳头干细胞(SCAPs)增殖及迁移能力起调节作用，为临床牙髓再生治疗提供新的理论基础。方法采用髓腔暴露法构建大鼠年轻恒牙根尖周炎模型，分离、培养鉴定并分为炎症环境中的SCAPs(AP-SCAPs)组和健康状态下SCAPs(H-SCAPs)组，采用CCK-8及划痕实验检测细胞增殖、迁移能力，q-PCR检测2组METTL3表达水平；慢病毒转染构建过表达METTL3的AP-SCAPs(Lv-M), 设置对照组Lv-M-Ctrl，siRNA干扰法构建沉默METTL3的AP-SCAPs(si-M)，对照组si-M-Ctrl，采用CCK-8和划痕实验检测各组细胞增殖、迁移能力。结果 AP-SCAPs较H-SCAPs的增殖、迁移能力降低(P＜0.05)，AP-SCAPs的METTL3表达量明显低于H-SCAPs(1.00±0.05 vs. 1.62±0.17, P=0.004)；CCK-8结果显示，Lv-M较对照组的增殖能力在24 h、72 h、120 h、168 h均增强(P＜0.05)；si-M与si-M-Ctrl相比，细胞增殖能力均下降(P＜0.05)。划痕实验结果显示，Lv-M组细胞迁移率明显高于Lv-M-Ctrl组[(70.21±3.66)% vs. (27.26±4.69)%, P＜0.001]；si-M组细胞迁移率较si-M-Ctrl组降低[(44.11±3.90)% vs. (72.33±5.61)%, P=0.002]。结论 METTL3可以调节炎症环境中SCAPs增殖和迁移的能力。
- 根尖牙乳头干细胞 /
- METTL3 /
- 增殖 /
- 迁移
Abstract: Objective To explore whether METTL3 plays a regulatory role in the proliferation and migration capacity of apical dental papilla stem cells (SCAPs) in inflammatory environments and to provide a new theoretical basis for clinical endodontic regenerative therapy. Methods A permanent tooth apical periodontitis model of young rat was constructed by means of the pulp exposure method. In addition, AP-SCAPs and H-SCAPs were isolated, cultured and identified. The proliferation and migration potential of the cells was detected by CCK-8 and scratch assay, and the expression level of METTL3 was detected by Q-PCR in the two groups. Lentiviral transfection was utilized to construct AP-SCAPs overex-pressing METTL3 (Lv-M), with Lv-M-Ctrl serving as the control. The construction of AP-SCAPs silencing METTL3 (si-M) was achieved by employing the group. siRNA interference method. The proliferation and migration ability of cells in each group was detected by CCK-8 and scratch assay, respectively. Results In comparison with H-SCAPs, AP-SCAPs demonstrated a reduced capacity for proliferation and migration (P < 0.05). Furthermore, and the METTL3 expression levels in AP-SCAPs were significantly lower than those in H-SCAPs (1.00±0.05 vs. 1.62±0.17, P=0.004). The CCK-8 results demonstrated that the proliferation capacity of Lv-M was elevated at 24, 72, 120 and 168 hours in comparison to the control group (P < 0.05). In comparison with the si-M-Ctrl group, the si-M group demonstrated a decrease in cell proliferation ability (P < 0.05). The results of the scratch assay demonstrated that the cell migration rate of the Lv-M group was higher than that of the Lv-M-Ctrl group [(70.21±3.66)% vs. (27.26±4.69)%, P < 0.001]. Furthermore, the cell migration rate of the si-M group was decreased in comparison with the si-M-Ctrl group [(44.11±3.90)% vs. (72.33±5.61)%, P=0.002]. Conclusion METTL3 has been demonstrated to play a regulatory role in the proliferation and migration of SCAPs within an inflammatory environment.
- Apical dental papilla stem cells /
- METTL3 /
- Proliferation /
- Migration

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图 1 SCAPs多向分化潜能鉴定(×100)

注：A、B为油红O染色结果；C、D为茜素红染色结果。

Figure 1. Identification of the multidirectional differentiation potential of SCAPs(×100)

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图 2 Western blotting验证过表达效果的蛋白表达条带图

Figure 2. Western blot analysis verifying overexpression protein (band diagram)

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图 3 Western blotting验证沉默效果的蛋白表达条带图

Figure 3. Western blot analysis verifying silencing protein (band diagram)

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表 1 实验所用引物序列

Table 1. Primer sequences used in this experiment

基因	引物	引物序列(5'-3')
METTL3	Forward	CCATCCGTCTTGCCATCTC
METTL3	Reverse	ATAGTCCCTGCTGCCTCTC
β-actin	Forward	CGGTCAGGTCATCACTATC
β-actin	Reverse	CAGGGCAGTAATCTCCTTC

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表 2 AP-SCAPs和H-SCAPs流式细胞术实验数据(x±s，%)

Table 2. Flow cytometry data of AP-SCAPs and H-SCAPs (x±s, %)

组别	n	CD29	CD34	CD45	CD90
AP-SCAPs	3	93.13±0.15	7.37±0.21	2.53±0.47	95.20±0.56
H-SCAPs	3	94.93±0.21	6.03±0.15	3.63±0.38	95.53±0.35

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表 3 AP-SCAPs和H-SCAPs CCK-8实验数据比较(x±s)

Table 3. Comparison of CCK-8 assay results for AP-SCAPs and H-SCAPs (x±s)

组别	n	0 h	24 h	72 h	120 h	168 h
AP-SCAPs	3	0.12±0.01	0.37±0.01	0.53±0.02	0.68±0.02	0.90±0.06
H-SCAPs	3	0.11±0.01	0.43±0.01	0.60±0.01	0.75±0.01	1.26±0.01
F值		1.124	17.064	7.082	5.991	10.963
P值		0.324	＜0.001	0.019	0.004	0.008

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表 4 Lv-M和Lv-M-Ctrl组CCK-8实验数据比较(x±s)

Table 4. Comparison of CCK-8 assay data for Lv-M and Lv-M Ctrl (x±s)

组别	n	0 h	24 h	72 h	120 h	168 h
Lv-M	3	0.16±0.01	0.36±0.01	0.52±0.01	0.72±0.01	0.88±0.01
Lv-M-Ctrl	3	0.13±0.01	0.31±0.01	0.47±0.01	0.64±0.01	0.75±0.01
F值		9.544	26.411	17.740	18.582	30.985
P值		＜0.001	＜0.001	＜0.001	＜0.001	＜0.001

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表 5 si-M和si-M-Ctrl CCK-8实验数据比较(x±s)

Table 5. Comparison of CCK-8 assay data for si-M and si-M-Ctrl (x±s)

组别	n	0 h	24 h	72 h	120 h	168 h
si-M	3	0.16±0.01	0.31±0.01	0.39±0.01	0.50±0.01	0.63±0.01
si-M-Ctrl	3	0.16±0.01	0.36±0.01	0.44±0.01	0.57±0.01	0.72±0.01
F值		0.234	8.847	13.587	23.031	22.508
P值		0.826	＜0.001	＜0.001	＜0.001	＜0.001

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