Zfp260对牙周炎破骨细胞分化影响的实验研究

doi:10.12439/kqhm.1005-4979.2025.02.002

摘要/Abstract

摘要：

目的： 探究锌指蛋白260（zinc finger protein 260，Zfp260）对牙周炎破骨细胞分化的影响。方法： 将Zfp260小干扰RNA（small interfering RNA，siRNA）转染进单核/巨噬细胞系RAW264.7，转染后的细胞在核因子κB受体活化因子配体（receptor activator of nuclear factor κB ligand，RANKL）诱导7 d后，通过抗酒石酸酸性磷酸酶（tartrate resistant acid phosphatase，TRAP）染色观察破骨细胞的形成，实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）检测破骨细胞分化标志基因的表达水平。基于Zfp260^flox/flox小鼠（对照组）及基因型为Zfp260^flox/flox Lyz2-cre^-的髓系细胞条件性敲除Zfp260基因（Zfp260 conditional knockout，Zfp260^CKO）小鼠（实验组）构建牙周炎模型，通过Micro-CT和组织学染色评价牙槽骨骨质破坏情况。结果： Zfp260在RAW264.7细胞向破骨细胞分化过程中表达上调；沉默Zfp260表达后，RAW264.7细胞破骨分化能力下降，破骨分化标志基因表达下调（P<0.05）；Zfp260^CKO组小鼠牙周炎侧骨吸收程度较轻。结论： Zfp260正向调控破骨细胞形成促进骨吸收，提示Zfp260可能成为牙周炎骨吸收的防治靶点。

关键词: 牙周炎, 牙槽骨吸收, 破骨细胞, 锌指蛋白260

Abstract:

Objective: To research the role of zinc finger protein 260 (Zfp260) in osteoclast differentiation under the periodontitis microenvironment.Methods: Small interfering RNA (siRNA) was transfected into the monocyte/macrophage cell line RAW264.7. The transfected cells were induced into osteoclasts by receptor activator of nuclear factor κB ligand (RANKL） for 7 days. Differentiated cells were harvested for tartrate resistant acid phosphatase (TRAP) staining. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression level of osteoclast differentiation-related genes. In vivo, experimental periodontitis models were established in Zfp260^flox/flox mice (Control group) and Zfp260^flox/flox Lyz2-cre^- myeloid cell conditional Zfp260-knockout mice (Zfp260^cko, Experimental group). The Micro-CT scan and histological staining were used to analyze alveolar bone resorption.Results: Zfp260 was up-regulated during osteoclast genesis. The knockdown of Zfp260 inhibited the differentiation of RAW264.7 cells into osteoclasts, and the expression of osteoclast-related factors was down-regulated (P<0.05). In vivo, the bone resorption was significantly decreased on the experimental side in Zfp260^CKO mice.Conclusion: Zfp260 regulates the osteoclast differentiation ability, which will provide a new target for solving periodontitis.

Key words: periodontitis, alveolar bone resorption, osteoclasts, zinc finger protein 260

中图分类号:

R782

贡若男, 王佐林. Zfp260对牙周炎破骨细胞分化影响的实验研究[J]. 《口腔颌面外科杂志》, 2025, 35(2): 91-97.

GONG Ruonan, WANG Zuolin. Experimental study of the effect of Zfp260 on the differentiation of osteoclasts in periodontitis[J]. 《Journal of Oral and Maxillofacial Surgery》, 2025, 35(2): 91-97.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2025/V35/I2/91

图/表 8

表1 引物序列

Table 1 Sequences of primers for RT-qPCR

引物名称	正向序列(5'-3')	反向序列(5'-3')
GAPDH	GGGAAGCCCATCACCATCTT	GCCTCACCCCATTTGATGTT
Zfp260	ATCTTCTGCATGACGAACCTG	GGCTTGTGAGGACTCTTCGC
CTSK	CAGCAGAGGTGTGTACTATG	GCGTTGTTCTTATTCCGAGC
TRAP	CACTCCCACCCTGAGATTTGT	CATCGTCTGCACGGTTCTG
MMP-9	TGAGTCCGGCAGACAATCCT	CCACGTGCGGGCAGTAA

图1 破骨诱导后相关基因的相对表达变化 A、B. 依次为RANKL诱导0、7 d时TRAP和Zfp260 mRNA的相对表达量，***表示P<0.001，与诱导0 d时比较；C、D. 依次为诱导0、7 d时镜下细胞TRAP染色图（×100），黑色箭头所指为破骨细胞。

Figure 1 Expression change of related genes after osteoclast induction

图2 Zfp260及破骨细胞相关基因的相对表达量 A—C. 依次为Zfp260及破骨细胞相关基因CTSK、MMP9在si-NC组与si-Zfp260组中的mRNA相对表达水平，*表示P<0.05，**表示P<0.01，***表示P<0.001，与si-NC组比较。

Figure 2 Relative expression levels of Zfp260 and osteoclast-related genes

图3 破骨细胞TRAP染色图及数量的统计学分析 A、B. 依次为si-NC组和si-Zfp260组向破骨诱导分化后的TRAP染色图（×100），黑色箭头所指为破骨细胞；C. 镜下TRAP阳性多核细胞的数量，**表示P<0.01，与si-NC组比较。

Figure 3 TRAP staining images and statistical analysis of osteoclast quantification

图4 2组小鼠下颌骨牙周炎模型三维重建图 Zfp260^flox/flox组和Zfp260^CKO组小鼠下颌骨三维重建图，从上至下分别为2组对照侧和实验侧的颊侧面及冠状面截图。

Figure 4 Three-dimensional reconstruction of mandibular periodontitis model in two groups of mice

图5 Micro-CT相关数据定量分析结果 A—E. 依次为2组小鼠实验侧和对照侧下颌骨BMD、Tb.N、Tb.Sp、Tb.Th、BV/TV数据的统计学分析图，*表示P<0.05，**表示P<0.01，与Zfp260^flox/flox组比较。

Figure 5 Quantitative analysis results of Micro-CT

图6 2组小鼠第一磨牙HE染色图 A—D. 依次为Zfp260^flox/flox组、Zfp260^CKO组小鼠对照侧、实验侧第一磨牙HE染色图（×40）；图中虚线a表示CEJ，虚线b表示ABC，a至b间的虚线为CEJ—ABC连线。

Figure 6 HE staining of the first molars of mice in the two groups

图7 2组小鼠下颌骨牙周炎组织TRAP染色图 A—D. 依次为Zfp260^flox/flox组、Zfp260^CKO组小鼠对照侧及实验侧第一磨牙TRAP染色图(×100)，每张的右侧图为左侧图矩形框内根分叉区域的放大图（×400）；E. 镜下TRAP阳性多核细胞数量统计分析图，*表示P<0.05，与Zfp260^flox/flox组比较。

Figure 7 TRAP staining images of periodontitis tissue in the mandible of mice in the two groups

参考文献 21

[1]	Becerra-Ruiz JS, Guerrero-Velázquez C, Martínez-Esquivias F, et al. Innate and adaptive immunity of periodontal disease. From etiology to alveolar bone loss[J]. Oral Dis, 2022, 28(6): 1441-1447.
[2]	Hajishengallis G. Periodontitis: From microbial immune subversion to systemic inflammation[J]. Nat Rev Immunol, 2015, 15(1): 30-44. doi: 10.1038/nri3785 pmid: 25534621
[3]	Khassawneh B, Alhabashneh R, Ibrahim F. The association between bronchial asthma and periodontitis: A case-control study in Jordan[J]. J Asthma, 2019, 56(4): 404-410. doi: 10.1080/02770903.2018.1466315 pmid: 29667525
[4]	Fatima Z, Shahzadi C, Nosheen A, et al. Periodontitis is a risk factor in developing cardiovascular diseases[J]. J Pak Med Assoc, 2020: 1-8.
[5]	Ono T, Nakashima T. Recent advances in osteoclast biology[J]. Histochem Cell Biol, 2018, 149(4): 325-341. doi: 10.1007/s00418-018-1636-2 pmid: 29392395
[6]	李琴, 王佐林. TREM-2/DAP12复合物在牙周炎中的作用及研究进展[J]. 口腔颌面外科杂志, 2021, 31(2): 110-113. doi: 10.3969/j.issn.1005-4979.2021.02.009
[7]	Tanaka Y, Nakayamada S, Okada Y. Osteoblasts and osteoclasts in bone remodeling and inflammation[J]. Curr Drug Target Inflamm Allergy, 2005, 4(3): 325-328.
[8]	Blottière L, Apiou F, Ferbus D, et al. Cloning, characterization, and chromosome assignment of Zfp146 the mouse ortholog of human ZNF146 a gene amplified and overexpressed in pancreatic cancer, and Zfp260 a closely related gene[J]. Cytogenet Cell Genet, 1999, 85(3-4): 297-300. pmid: 10449921
[9]	Chen K, Zhang N, Shao JB, et al. Allogeneic hematopoietic stem cell transplantation for PEX1-related zellweger spectrum disorder: A case report and literature review[J]. Front Pediatr, 2021, 9: 672187.
[10]	Komati H, Maharsy W, Beauregard J, et al. ZFP260 is an inducer of cardiac hypertrophy and a nuclear mediator of endothelin-1 signaling[J]. J Biol Chem, 2011, 286(2): 1508-1516. doi: 10.1074/jbc.M110.162966 pmid: 21051538
[11]	Jurado Acosta A, Rysä J, Szabo Z, et al. Transcription factor PEX1 modulates extracellular matrix turnover through regulation of MMP-9 expression[J]. Cell Tissue Res, 2017, 367(2): 369-385. doi: 10.1007/s00441-016-2527-2 pmid: 27826738
[12]	Li J, Jin F, Cai M, et al. LncRNA nron inhibits bone resorption in periodontitis[J]. J Dent Res, 2022, 101(2): 187-195.
[13]	Kim JM, Lin CJ, Stavre Z, et al. Osteoblast-osteoclast communication and bone homeostasis[J]. Cells, 2020, 9(9): 2073.
[14]	Uenaka M, Yamashita E, Kikuta J, et al. Osteoblast-derived vesicles induce a switch from bone-formation to bone-resorption in vivo[J]. Nat Commun, 2022, 13(1): 1066. doi: 10.1038/s41467-022-28673-2 pmid: 35210428
[15]	Chen X, Wang ZQ, Duan N, et al. Osteoblast-osteoclast interactions[J]. Connect Tissue Res, 2018, 59(2): 99-107. doi: 10.1080/03008207.2017.1290085 pmid: 28324674
[16]	Li WJ, Xie HL, Hu H, et al. PEX1 is a mediator of α1-adrenergic signaling attenuating doxorubicin-induced cardiotoxicity[J]. J Biochem Mol Toxicol, 2022, 36(11): e23196.
[17]	Debrus S, Rahbani L, Marttila M, et al. The zinc finger-only protein Zfp260 is a novel cardiac regulator and a nuclear effector of alpha1-adrenergic signaling[J]. Mol Cell Biol, 2005, 25(19): 8669-8682. pmid: 16166646
[18]	Jurado Acosta A, Rysä J, Szabo Z, et al. Transcription factor PEX1 modulates extracellular matrix turnover through regulation of MMP-9 expression[J]. Cell Tissue Res, 2017, 367(2): 369-385. doi: 10.1007/s00441-016-2527-2 pmid: 27826738
[19]	Kaur S, Bansal Y, Kumar R, et al. A panoramic review of IL-6: Structure, pathophysiological roles and inhibitors[J]. Bioorg Med Chem, 2020, 28(5): 115327.
[20]	Garbers C, Aparicio-Siegmund S, Rose-John S. The IL-6/gp130/STAT3 signaling axis: Recent advances towards specific inhibition[J]. Curr Opin Immunol, 2015, 34: 75-82. doi: 10.1016/j.coi.2015.02.008 pmid: 25749511
[21]	Kang WY, Shang LL, Wang T, et al. Rho-kinase inhibitor Y-27632 downregulates LPS-induced IL-6 and IL-8 production via blocking p38 MAPK and NF-κB pathways in human gingival fibroblasts[J]. J Periodontol, 2018, 89(7): 883-893. doi: 10.1002/JPER.17-0571 pmid: 29630729

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