NAT10对小鼠拔牙创愈合影响的实验研究

doi:10.12439/kqhm.1005-4979.2026.05.002

摘要/Abstract

摘要：

目的：

探究N-乙酰转移酶10（N-acetyltransferase 10，NAT10）对小鼠拔牙创愈合的影响。

方法：

建立小鼠拔牙创愈合模型，检测不同时间点拔牙创组织中NAT10的表达水平。将48只雄性C57BL/6小鼠随机分为实验组和对照组，建立拔牙创模型后分别局部注射NAT10抑制剂Remodelin（实验组）和赋形剂（对照组）。利用苏木精-伊红（hematoxylin and eosin，HE）染色、实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）、免疫荧光染色及Micro-CT检测2组小鼠拔牙创愈合情况。体外分离培养颌骨骨髓间充质干细胞（jaw bone marrow mesenchymal stem cells，JBMMSCs），使用Remodelin抑制NAT10表达。通过RT-qPCR、蛋白质印迹法、碱性磷酸酶（alkaline phosphatase，ALP）染色、茜素红S（alizarin red S，ARS）染色评价JBMMSCs成骨分化能力。

结果：

HE染色结果显示，拔牙后第7天拔牙创内开始形成新生骨组织，第14~21天发生骨基质矿化及骨改建。RT-qPCR结果显示，NAT10在拔牙后第3 天表达水平最高。Remodelin处理后，实验组拔牙创组织中NAT10表达明显降低；免疫荧光染色、HE染色及Micro-CT结果显示，与对照组相比，实验组RUNX2阳性细胞减少，新生骨形成下降，骨体积分数（bone volume fraction，BV/TV）及骨小梁厚度（trabecular thickness，Tb.Th）降低，拔牙创骨愈合延迟。体外实验结果显示，抑制NAT10表达后，JBMMSCs成骨相关基因及蛋白表达下调，ALP染色和ARS染色强度减弱，成骨分化能力降低。

结论：

NAT10一定程度上维持了JBMMSCs的成骨分化能力，有利于小鼠拔牙创的骨愈合。

关键词: N-乙酰转移酶10, 拔牙创愈合, 颌骨骨髓间充质干细胞

Abstract:

Objective:

To investigate the effect of N-acetyltransferase 10 (NAT10) on the healing of tooth extraction sockets in mice.

Methods:

A mouse model of tooth extraction socket healing was established, and the expression of NAT10 in extraction socket tissues at different time points was evaluated. A total of 48 male C57BL/6 mice were randomly assigned to an experimental group and a control group. Following tooth extraction, the experimental group received local injection of the NAT10 inhibitor Remodelin, whereas the control group received vehicle treatment. Hematoxylin and eosin (HE) staining, real-time quantitative polymerase chain reaction (RT-qPCR), immunofluorescence staining, and Micro-CT were performed to evaluate extraction socket healing. Jaw bone marrow mesenchymal stem cells (JBMMSCs) were isolated and cultured in vitro. NAT10 expression was inhibited using Remodelin, and the effects on osteogenic differentiation were assessed by RT-qPCR, Western blotting, alkaline phosphatase (ALP) staining, and alizarin red S (ARS) staining.

Results:

HE staining demonstrated that newly formed bone appeared in the extraction sockets on day 7 after tooth extraction, whereas bone matrix mineralization and bone remodeling occurred from day 14 to day 21. RT-qPCR analysis showed that NAT10 expression reached its peak on day 3 after tooth extraction. Following Remodelin treatment, NAT10 expression in extraction socket tissues was markedly reduced. Immunofluorescence staining, HE staining, and Micro-CT analyses revealed that, compared with the control group, the experimental group exhibited fewer RUNX2-positive cells, reduced new bone formation, decreased bone volume fraction (BV/TV) and trabecular thickness (Tb.Th), and delayed extraction socket bone healing. In vitro experiments showed that inhibition of NAT10 downregulated the expression of osteogenesis-related genes and proteins in JBMMSCs, reduced ALP and ARS staining intensity, and impaired osteogenic differentiation.

Conclusion:

NAT10 maintained the osteogenic differentiation ability of JBMMSCs to some extent, and was beneficial to bone healing after tooth extraction in mice.

Key words: N-acetyltransferase 10, tooth extraction socket healing, jaw bone marrow mesenchymal stem cells

中图分类号:

R782.1

杨东荣, 陈永亮, 王菁, 王海丞, 王佐林. NAT10对小鼠拔牙创愈合影响的实验研究[J]. 《口腔颌面外科杂志》, 2026, 36(5): 367-375.

YANG Dongrong, CHEN Yongliang, WANG Jing, WANG Haicheng, WANG Zuolin. Effect of NAT10 on the healing of tooth extraction sockets in mice[J]. Journal of Oral and Maxillofacial Surgery(CN), 2026, 36(5): 367-375.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2026/V36/I5/367

图/表 8

表1 RT-qPCR引物序列

Table 1 Primer sequences used for RT-qPCR

基因名	正向引物序列（5'-3'）	反向引物序列（5'-3'）
GAPDH	AGGTCGGTGTGAACGGATTTG	TGTAGACCATGTAGTTGAGGTCA
NAT10	GAGCTGGGATTTAGCAGTCAC	CAGTAGCGAATGTTTGTGGCT
Runx2	CCCAGCCACCTTTACCTACA	TATGGAGTGCTGCTGGTCTG
Alp	GCTGATCATTCCCACGTTTT	CTGGGCCTGGTAGTTGTTGT
Bglap	TTTCTGCTCACTCTGCTGACC	GCTTGGACATGAAGGCTTTGT
Sp7	ATGGCGTCCTCTCTGCTTG	TGAAAGGTCAGCGTATGGCTT

图1 小鼠拔牙创愈合过程中组织学变化及NAT10表达 A.建模后3、7、14、21 d拔牙创HE染色结果，白色虚线指示拔牙创部位（×10）；B. NAT10在拔牙创愈合过程中的mRNA相对表达水平，**表示P<0.01，与第0天时比较。

Figure 1 Histological changes and NAT10 expression during tooth extraction socket healing in mice

图2 Remodelin对拔牙创中NAT10表达的抑制作用 A.建模第3天对照组与实验组NAT10免疫荧光染色结果，蓝色示细胞核，绿色示NAT10，白色箭头示表达NAT10的细胞（×20）；B.建模第7天RT-qPCR检测拔牙创组织NAT10 mRNA相对表达量。*表示P<0.05，与对照组比较。

Figure 2 Inhibitory effect of Remodelin on NAT10 expression in extraction sockets

图3 抑制NAT10对拔牙创骨愈合的影响 A.建模后5 d，实验组与对照组免疫荧光染色结果，右侧2列（×20）为左侧（×10）白色矩形区域的放大图，白色箭头示表达RUNX2的细胞；B.建模5 d后的RUNX2阳性细胞定量分析；C.建模后14 d对照组与实验组拔牙创Micro-CT图像（冠状位）及HE染色图（×10），图中黄色虚线示拔牙创；D.建模14 d后BV/TV与Tb.Th的定量分析结果。**表示P<0.01，***表示P<0.001，与对照组比较。

Figure 3 Effect of NAT10 inhibition on extraction socket bone healing

图4 Micro-CT检测建模后14 d空白组与对照组BV/TV、Tb.Th

Figure 4 Micro-CT analysis of BV/TV and Tb.Th in the untreated and control groups 14 days after modeling

图5 JBMMSCs的分离与鉴定 A.第3代JBMMSCs的形态（×10）；B、C. CD29、CD44呈阳性表达；D—F. CD11b、CD31、CD45呈阴性表达。

Figure 5 Isolation and identification of JBMMSCs

图6 Remodelin对JBMMSCs中NAT10表达的抑制作用 A.各组蛋白条带图；B. RT-qPCR结果及蛋白质定量分析，*表示P<0.05，**表示P<0.01，与对照组比较。

Figure 6 Inhibitory effect of Remodelin on NAT10 expression in JBMMSCs

图7 抑制NAT10对JBMMSCs成骨分化的影响 A.成骨相关基因RT-qPCR检测结果；B、C. 2组蛋白条带图及蛋白相对表达量定量分析；D、E. ALP及ARS染色结果。*表示P<0.05，**表示P<0.01，***表示P<0.001，与对照组比较。

Figure 7 Effect of NAT10 inhibition on osteogenic differentiation of JBMMSCs

参考文献 30

[1]	Marquezan M, Osório A, Sant'Anna E, et al. Does bone mineral density influence the primary stability of dental implants? A systematic review[J]. Clin Oral Implants Res, 2012, 23(7): 767-774.
[2]	郭向飞, 郝颖, 赵烨, 等. N-乙酰基转移酶10的研究进展[J]. 生命的化学, 2020, 40(11): 2014-2019.
	Guo XF, Hao Y, Zhao Y, et al. Research advances on the function of N-acetyltransferase 10[J]. Chem Life, 2020, 40(11): 2014-2019.
[3]	Yang W, Li HY, Wu YF, et al. ac4C acetylation of RUNX2 catalyzed by NAT10 spurs osteogenesis of BMSCs and prevents ovariectomy-induced bone loss[J]. Mol Ther Nucleic Acids, 2021, 26: 135-147.
[4]	王姗. NAT10在胰腺癌细胞恶性生物学行为中的作用及初步机制研究[D]. 长沙: 中南大学, 2022.
	Wang S. The role and preliminary mechanism of NAT10 in malignant biological behaviors of pancreatic cancer cells[D]. Changsha: Central South University, 2022.
[5]	Aghaloo TL, Chaichanasakul T, Bezouglaia O, et al. Osteogenic potential of mandibular vs. long-bone marrow stromal cells[J]. J Dent Res, 2010, 89(11): 1293-1298.
[6]	Dong W J, Ge J, Zhang P, et al. Phenotypic characterization of craniofacial bone marrow stromal cells: Unique properties of enhanced osteogenesis, cell recruitment, autophagy, and apoptosis resistance[J]. Cell Tissue Res, 2014, 358(1): 165-175.
[7]	Akintoye SO. The distinctive jaw and alveolar bone regeneration[J]. Oral Dis, 2018, 24(1/2): 49-51.
[8]	Ukwas A, Elbego A, Albego M, et al. Non-healing extraction socket: A diagnostic challenge for general dental practitioners[J]. Br Dent J, 2024, 237(12): 911-916.
[9]	Yuan X, Pei X, Zhao Y, et al. A wnt-responsive PDL population effectuates extraction socket healing[J]. J Dent Res, 2018, 97(7): 803-809.
[10]	Cui Y, Hong SB, Xia YH, et al. Melatonin engineering M2 macrophage-derived exosomes mediate endoplasmic reticulum stress and immune reprogramming for periodontitis therapy[J]. Adv Sci (Weinh), 2023, 10(27): e2302029.
[11]	Nisar N, Nilesh K, Parkar MI, et al. Extraction socket preservation using a collagen plug combined with platelet-rich plasma (PRP): A comparative clinico-radiographic study[J]. J Dent Res Dent Clin Dent Prospects, 2020, 14(2): 139-145.
[12]	Wang Y, Fang JK, Liu BM, et al. Reciprocal regulation of mesenchymal stem cells and immune responses[J]. Cell Stem Cell, 2022, 29(11): 1515-1530.
[13]	Zhang D, Zhang S, Wang J, et al. LepR-expressing stem cells are essential for alveolar bone regeneration[J]. J Dent Res, 2020, 99(11): 1279-1286.
[14]	Jeffery EC, Mann TLA, Pool JA, et al. Bone marrow and periosteal skeletal stem/progenitor cells make distinct contributions to bone maintenance and repair[J]. Cell Stem Cell, 2022, 29(11): 1547-1561.e6.
[15]	Wang GP, Zhang M, Zhang YM, et al. NAT10-mediated mRNA N4-acetylcytidine modification promotes bladder cancer progression[J]. Clin Transl Med, 2022, 12(5): e738.
[16]	Yang CX, Wu TT, Zhang J, et al. Prognostic and immunological role of mRNA ac4C regulator NAT10 in pan-cancer: New territory for cancer research?[J]. Front Oncol, 2021, 11: 630417.
[17]	Jin GH, Xu MQ, Zou MS, et al. The processing, gene regulation, biological functions, and clinical relevance of N4-acetylcytidine on RNA: A systematic review[J]. Mol Ther Nucleic Acids, 2020, 20: 13-24.
[18]	Dominissini D, Rechavi G. N4-acetylation of cytidine in mRNA by NAT10 regulates stability and translation[J]. Cell, 2018, 175(7): 1725-1727.
[19]	Sleiman S, Dragon F. Recent advances on the structure and function of RNA acetyltransferase Kre33/NAT10[J]. Cells, 2019, 8(9): 1035.
[20]	Larrieu D, Britton S, Demir M, et al. Chemical inhibition of NAT10 corrects defects of laminopathic cells[J]. Science, 2014, 344(6183): 527-532.
[21]	王凯, 宋敏, 文皓楠, 等. 转化生长因子-β在骨代谢中作用机制的研究进展[J]. 中国骨质疏松杂志, 2020, 26(2): 308-312.
	Wang K, Song M, Wen HN, et al. Research progress in the mechanism of action of transforming growth factor-β in bone metabolism[J]. Chin J Osteoporos, 2020, 26(2): 308-312.
[22]	Xu D, Huang KG, Chen Y, et al. Immune response and drug therapy based on ac4C-modified gene in pancreatic cancer typing[J]. Front Immunol, 2023, 14: 1133166.
[23]	Onuora S. Tenascin C promotes pathological bone formation in AS[J]. Nat Rev Rheumatol, 2021, 17(7): 379.
[24]	Ikeuchi Y, Kitahara K, Suzuki T. The RNA acetyltransferase driven by ATP hydrolysis synthesizes N4-acetylcytidine of tRNA anticodon[J]. EMBO J, 2008, 27(16): 2194-2203.
[25]	Kusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone[J]. Nature, 2014, 507(7492): 323-328.
[26]	Hu YJ, Wu HT, Xu T, et al. Defactinib attenuates osteoarthritis by inhibiting positive feedback loop between H-type vessels and MSCs in subchondral bone[J]. J Orthop Translat, 2020, 24: 12-22.
[27]	Lu JS, Zhang HY, Cai DZ, et al. Positive-feedback regulation of subchondral H-type vessel formation by chondrocyte promotes osteoarthritis development in mice[J]. J Bone Miner Res, 2018, 33(5): 909-920.
[28]	Wu HT, Xu T, Chen ZG, et al. Specific inhibition of FAK signaling attenuates subchondral bone deterioration and articular cartilage degeneration during osteoarthritis pathogenesis[J]. J Cell Physiol, 2020, 235(11): 8653-8666.
[29]	Zafari F, Shirian S, Sadeghi M, et al. CD93 hematopoietic stem cells improve diabetic wound healing by VEGF activation and downregulation of DAPK-1[J]. J Cell Physiol, 2020, 235(3): 2366-2376.
[30]	Fei XF, Cai YY, Lin F, et al. Amniotic fluid mesenchymal stem cells repair mouse corneal cold injury by promoting mRNA N4-acetylcytidine modification and ETV4/JUN/CCND2 signal axis activation[J]. Hum Cell, 2021, 34(1): 86-98.

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