去铁胺局部注射对小鼠牙槽骨骨改建的实验研究

doi:10.12439/kqhm.1005-4979.2024.02.002

摘要/Abstract

摘要：

目的： 探究局部注射去铁胺（desferrioxamine，DFO）通过低氧诱导因子-1α（hypoxia inducible factor-1α，HIF-1α）调控破骨细胞对牙槽骨骨改建的影响。方法： 对C57小鼠局部牙槽骨注射DFO，通过micro-CT及组织学染色法观察牙槽骨骨量的变化；通过免疫荧光及抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphase，TRAP)染色观察破骨细胞生成，之后用TRAP、免疫荧光等方法观察体外DFO通过HIF-1α对Raw264.7向破骨细胞分化的影响。结果： Micro-CT及组织学染色显示，DFO局部注射后局部骨密度降低、骨量减少；组织学染色发现，相比对照组，DFO注射组局部破骨细胞数量增多、HIF-1α表达增加；体外细胞培养发现DFO可促进Raw264.7细胞中HIF-1α的表达及其向破骨细胞分化。结论： DFO局部注射可促进牙槽骨骨改建及Raw264.7向破骨细胞分化。

关键词: 破骨细胞, 低氧诱导因子-1α, 牙槽骨

Abstract:

Objective: To investigate the effect of local injection of desferrioxamine (DFO) on the bone remodeling of alveolar bone through the regulation of osteoclasts by hypoxia inducible factor-1α (HIF-1α). Methods: DFO was injected locally into the alveolar bone of C57 mice; changes in alveolar bone volume were observed by micro-CT and histological staining; osteoclastogenesis was observed by immunofluorescence and tartrate-resistant acid phosphase (TRAP) staining; the effects of DFO on the differentiation of Raw264.7 to osteoclasts via HIF-1α were observed in vitro by TRAP staining and immunofluorescence. Results: Micro-CT and histological staining showed that local bone density decreased and bone volume decreased after local injection of DFO; histological staining revealed that the number of local osteoclasts increased and HIF-1α expression increased in the DFO injection group compared with the control group; in vitro cell culture revealed that DFO promoted the expression of HIF-1α in Raw264.7 cells and its differentiation to osteoclasts. Conclusion: Local injection of DFO can promote bone remodeling of alveolar bone and differentiation of Raw264.7 into osteoclasts.

Key words: osteoclasts, hypoxia-inducible factor-1α, alveolar bone

中图分类号:

R782

张倩, 康非吾. 去铁胺局部注射对小鼠牙槽骨骨改建的实验研究[J]. 《口腔颌面外科杂志》, 2024, 34(2): 87-93.

ZHANG Qian, KANG Feiwu. Local injection of DFO on bone remodeling of alveolar bone: An experimental study in the mouse[J]. 《Journal of Oral and Maxillofacial Surgery》, 2024, 34(2): 87-93.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2024/V34/I2/87

图/表 4

表1 RT-qPCR引物序列

Table 1 Primer sequences used for RT-qPCR

基因	正向序列(5'-3')	反向序列(5'-3')
HIF-1α	GAATGAAGTGCACCCTAACAAG	GAGGAATGGGTTCACAAATCAG
β-actin	GAAATCGTGCGTGACATCAAA	TGTAGTTTCATGGATGCCACAG

图1 小鼠上颌骨第一、二磨牙间牙槽骨骨密度变化 A.X线片、micro-CT扫描及组织三维重建，红色虚线区域为图像分析的感兴趣区域（area of interest，AOI）；B.A图的统计学分析，测量值包括BV/TV、Tb.N、Tb.Sp、Conn.D，*表示P<0.05，**表示P<0.01，与对照组比较；C.小鼠上颌骨第一、二磨牙间牙槽骨HE染色、MASSON染色（×40，×200）。

Figure 1 Changes in bone density of alveolar bone between the first and second molars in the maxilla of mice

图2 小鼠上颌骨第一、二磨牙间破骨细胞数目变化 A.小鼠上颌骨第一、二磨牙间牙槽骨HIF-1α和CTSK双重免疫荧光染色（×200，×400），白色箭头所指为多核破骨细胞；B.TRAP染色结果（×200，×400）；C.HIF-1α和CTSK双重免疫荧光染色统计学分析；D.TRAP染色统计学分析。*表示P<0.05，**表示P<0.01，***表示P<0.001，与对照组比较。

Figure 2 Changes in the number of osteoclasts between the first and second molars in the maxilla of mice

图3 DFO对RAW264.7细胞系破骨向分化的影响 A.CCK-8检测DFO对细胞活力的影响；B.RT-qPCR实验检测破骨细胞HIF-1α mRNA的相对表达量（以β-actin为内参）；C.细胞免疫荧光实验（×200）检测HIF-1α、CTSK蛋白的表达；D.TRAP染色结果（×200）；E、F.D图TRAP染色的统计学分析。**表示P<0.01，***表示P<0.001，与对照组比较。

Figure 3 Effects of DFO on osteoblastic differentiation of RAW264.7 cell

参考文献 20

[1]	Keim RG, Gottlieb EL, Nelson AH, et al. 2013 JCO orthodontic practice study. Part 1: Trends[J]. J Clin Orthod, 2013, 47(11): 661-680.
[2]	Ong MM, Wang HL. Periodontic and orthodontic treatment in adults[J]. Am J Orthod Dentofacial Orthop, 2002, 122(4): 420-428. doi: 10.1067/mod.2002.126597 URL
[3]	Wilcko WM, Wilcko T, Bouquot JE, et al. Rapid orthodontics with alveolar reshaping: Two case reports of decrowding[J]. Int J Periodontics Restorative Dent, 2001, 21(1): 9-19.
[4]	蔡昀, 唐燚, 康非吾. 下颌升支截骨去血供后牙槽骨内氧水平变化与骨改建的变化研究[J]. 口腔医学, 2020, 40(10): 869-873.
[5]	Ballas SK, Zeidan AM, Duong VH, et al. The effect of iron chelation therapy on overall survival in sickle cell disease and β-thalassemia: A systematic review[J]. Am J Hematol, 2018, 93(7): 943-952. doi: 10.1002/ajh.25103 pmid: 29635754
[6]	Zeng YW, Huang C, Duan DM, et al. Injectable temperature-sensitive hydrogel system incorporating deferoxamine-loaded microspheres promotes H-type blood vessel-related bone repair of a critical size femoral defect[J]. Acta Biomater, 2022, 153: 108-123. doi: 10.1016/j.actbio.2022.09.018 URL
[7]	Semenza GL. Hypoxia-inducible factors in physiology and medicine[J]. Cell, 2012, 148(3): 399-408. doi: 10.1016/j.cell.2012.01.021 pmid: 22304911
[8]	Tang Y, Zhu J, Huang DQ, et al. Mandibular osteotomy-induced hypoxia enhances osteoclast activation and acid secretion by increasing glycolysis[J]. J Cell Physiol, 2019, 234(7): 11165-11175. doi: 10.1002/jcp.27765 pmid: 30548595
[9]	Xue YR, Zhang GJ, Zhou SJ, et al. Iron chelator induces apoptosis in osteosarcoma cells by disrupting intracellular iron homeostasis and activating the MAPK pathway[J]. Int J Mol Sci, 2021, 22(13): 7168. doi: 10.3390/ijms22137168 URL
[10]	Li LH, Yin XJ, Ma N, et al. Desferrioxamine regulates HIF-1 alpha expression in neonatal rat brain after hypoxia-ischemia[J]. Am J Transl Res, 2014, 6(4): 377-383. pmid: 25075254
[11]	Wu YC, Li XQ, Xie WJ, et al. Neuroprotection of deferoxamine on rotenone-induced injury via accumulation of HIF-1 alpha and induction of autophagy in SH-SY5Y cells[J]. Neurochem Int, 2010, 57(3): 198-205. doi: 10.1016/j.neuint.2010.05.008 pmid: 20546814
[12]	Jing XZ, Du T, Yang XX, et al. Desferoxamine protects against glucocorticoid-induced osteonecrosis of the femoral head via activating HIF-1α expression[J]. J Cell Physiol, 2020, 235(12): 9864-9875. doi: 10.1002/jcp.29799 pmid: 32437020
[13]	Gonzalez FJ, Xie C, Jiang CT. The role of hypoxia-inducible factors in metabolic diseases[J]. Nat Rev Endocrinol, 2018, 15(1): 21-32. doi: 10.1038/s41574-018-0096-z pmid: 30275460
[14]	Guan SY, Leng RX, Tao JH, et al. Hypoxia-inducible factor-1α: A promising therapeutic target for autoimmune diseases[J]. Expert Opin Ther Targets, 2017, 21(7): 715-723. doi: 10.1080/14728222.2017.1336539 URL
[15]	Li L, Qu Y, Jin X, et al. Protective effect of salidroside against bone loss via hypoxia-inducible factor-1α pathway-induced angiogenesis[J]. Sci Rep, 2016, 6: 32131. doi: 10.1038/srep32131 pmid: 27558909
[16]	Liberti MV, Locasale JW. The Warburg effect: How does it benefit cancer cells?[J]. Trends Biochem Sci, 2016, 41(3): 211-218. doi: S0968-0004(15)00241-8 pmid: 26778478
[17]	Zhu J, Tang Y, Wu Q, et al. HIF-1α facilitates osteocyte-mediated osteoclastogenesis by activating JAK2/STAT3 pathway in vitro[J]. J Cell Physiol, 2019, 234(11): 21182-21192. doi: 10.1002/jcp.28721 pmid: 31032948
[18]	Zhang XY, Cao JB, Zhang LM, et al. Deferoxamine attenuates lipopolysaccharide-induced neuroinflammation and memory impairment in mice[J]. J Neuroinflammation, 2015, 12: 20. doi: 10.1186/s12974-015-0238-3 URL
[19]	Pohl Y, Filippi A, Kirschner H. Results after replantation of avulsed permanent teeth. II. Periodontal healing and the role of physiologic storage and antiresorptive-regenerative therapy[J]. Dent Traumatol, 2005, 21(2): 93-101. doi: 10.1111/j.1600-9657.2004.00298.x pmid: 15773888
[20]	Kim JM, Lin CJ, Stavre Z, et al. Osteoblast-osteoclast communication and bone homeostasis[J]. Cells, 2020, 9(9): 2073. doi: 10.3390/cells9092073 URL

选择文件类型/文献管理软件名称

选择包含的内容