铜离子对牙髓组织再生能力的影响及其机制初探

doi:10.13591/j.cnki.kqyx.2023.05.004

摘要/Abstract

摘要：

目的研究铜离子对牙髓干细胞(dental pulp stem cells, DPSCs)的牙髓组织再生能力的影响及其机制。方法用不同浓度的铜离子刺激DPSCs,CCK-8检测铜离子对细胞的增殖和毒性作用;活死染色检测水凝胶毒性;Western blotting和qPCR检测低氧诱导因子1α(hypoxia inducible factor-1α, HIF-1α)、色素框同源蛋白7(chromobox protein homolog 7, CBX7)以及干细胞特性相关指标的蛋白和mRNA表达情况;人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)成管和迁移实验观察血管新生表型;制备负载DPSCs的含铜离子水凝胶并注射至裸鼠皮下,于术后14 d取材,应用苏木素-伊红和免疫组织化学染色观察细胞在裸鼠皮下的自我更新和血管新生情况。结果体外结果显示5、25、50 μmol/L铜离子能促进DPSCs增殖,对DPSCs无显著毒性作用;Western blotting和qPCR结果显示25 μmol/L铜离子组的HIF-1α、CBX7、Sox-2、Oct-4和Nanog的蛋白和mRNA的表达水平显著高于对照组;在HUVECs成管和迁移实验中25 μmol/L铜离子能促进HUVECs的成管和迁移。体内结果显示负载DPSCs的含铜离子水凝胶可以在裸鼠皮下诱导新生血管样组织,显著增加Sox-2阳性细胞的比例。结论铜离子能刺激DPSCs活化乏氧信号HIF-1α,增加CBX7表达,进而上调Sox-2、Oct-4、Nanog,维持DPSCs干细胞特性,同时能诱导血管新生,从而为实现牙髓组织再生提供理论依据和实验基础。

关键词: 铜离子, 牙髓干细胞, 牙髓再生, CBX7

Abstract:

Objective To study the effect of copper ions on the dental pulp regenerative ability of dental pulp stem cells (DPSCs) and its primary mechanism. Methods After DPSCs were stimulated with culture medium containing copper in different concentrations, CCK-8 assay was employed for evaluating the proliferation of DPSCs and toxic effects on DPSCs; Calcein-AM/PI kit was used to test the cytotoxicity of hydrogel; Western blotting and qPCR was used to detect the protein and mRNA expression of hypoxia inducible factor-1α (HIF-1α), chromobox protein homolog 7 (CBX7) and stemness-related indicators; the phenotype of angiogenesis was examined by human umbilical vein endothelial cells (HUVECs) tubes formation assay and migration assay; the copper-containing hydrogel loaded with DPSCs was prepared and injected into the subcutaneous of nude mice for two weeks, and then hematoxylin-eosin (HE) and immunohistochemistry (IHC) staining were used to observe the self-renewal and angiogenesis of DPSCs under the skin. Results The in vitro results showed that 5, 25, 50 μmol/L copper ions could promote the proliferation of DPSCs and had no significant toxic effects on DPSCs; Western blotting and qPCR results displayed the protein and mRNA levels of HIF-1α, CBX7, Sox-2, Oct-4 and Nanog in 25 μmol/L copper ions group were higher than that in the control group. Copper ions (25 μmol/L) could improve the tube formation and migration of HUVECs. The in vivo results showed that the copper-containing hydrogel loaded with DPSCs could lead to angiogenesis and increase dramatically the expression of Sox-2 in the subcutaneous of nude mice. Conclusion Copper ions can activate HIF-1α, enhance the expression of CBX7, and then upregulate Sox-2, Oct-4 and Nanog to maintain the stemness of DPSCs, meanwhile, can induce the regeneration of blood vessels, which can provide theoretical and experimental foundation for the regeneration of dental pulp.

Key words: copper ions, dental pulp stem cells, dental pulp regeneration, chromobox protein homolog 7(CBX7)

中图分类号:

R781.3

伍玉,杨磊婷,江飞,周芷萱,沈铭. 铜离子对牙髓组织再生能力的影响及其机制初探[J]. 口腔医学, 2023, 43(5): 407-414.

WU Yu,YANG Leiting,JIANG Fei,ZHOU Zhixuan,SHEN Ming. The effect and primary mechanism of copper ions on the regenerative capability of dental pulp[J]. Stomatology, 2023, 43(5): 407-414.

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引物名称		引物序列
hypoxia inducible factor-1α(HIF-1α)Forward:5'-TTCACCTGAGCCTAATAGTCC-3'		Reverse:5'-CAAGTCTAAATCTGTGTCCTG-3'
chromobox protein homolog 7(CBX7)Forward:5'- GGATGGCCCCCAAAGTACAG-3'		Reverse:5'- TATACCCCGATGCTCGGTCTC-3'
SRY-box transcription factor 2(Sox-2)Forward:5'-GGCAGAGAAGAGAGTGTTTGC-3'		Reverse:5'-GCCGCCGATGATTGTTATT-3'
Octamer-binding transcription factor 4(Oct-4)Forward:5'-TGAGAGGCAACCTGGAGAAT-3'		Reverse:5'-AACCACACTCGGACCACATC-3'
Nanog homeobox(Nanog)Forward:5'-AAGGTCCCGGTCAAGAAACAG-3'		Reverse:5'- CTTCTGCGTCACACCATTGC-3'
vascular endothelial growth factor A(VEGFA)Forward:5'-AGGGCAGAATCATCACGAAGT-3'		Reverse:5'-AGGGTCTCGATTGGATGGCA-3'
β-ActinForward:5'-TCATGAAGTGTGACGTGGACAT-3'		Reverse:5'-CTCAGGAGGAGCAATGATCTTG-3'