神经表皮生长因子样蛋白1对人脱落乳牙牙髓干细胞增殖和骨向分化的影响

doi:10.13591/j.cnki.kqyx.2025.07.007

摘要/Abstract

摘要：

目的探究神经表皮生长因子样蛋白1(Nel-like type 1 molecule,NELL-1)对人脱落乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth,SHEDs)增殖及骨向分化的影响。方法对SHEDs进行分离、培养和细胞鉴定,将生长状态良好的第3代细胞用于后续实验。SHEDs分为4组,分别加入浓度为0(对照组)、50、100、200 ng/mL的NELL-1,利用CCK-8法和结晶紫染色法检测细胞活性;碱性磷酸酶活性检测细胞成骨能力改变;RT-qPCR检测成骨相关基因ALP、OCN、Runx-2表达水平;Western blot法检测成骨相关蛋白ALP、Runx-2、Col-Ⅰ表达情况。结果 SHEDs具有干细胞特性,50 ng/mL的NELL-1可显著促进SHEDs增殖(P<0.01);碱性磷酸酶活性检测结果显示,加入NELL-1后,各组细胞成骨效果优于对照组,其中50 ng/mL NELL-1组成骨效果最佳(P<0.05);RT-qPCR和Western blot结果显示50 ng/mL NELL-1显著促进ALP、OCN、Runx-2成骨相关基因及ALP、Runx-2、Col-Ⅰ蛋白表达(P<0.05)。结论 NELL-1对SHEDs增殖、成骨分化具有促进作用。

关键词: 神经表皮生长因子样蛋白1, 人脱落乳牙牙髓干细胞, 成骨分化

Abstract:

Objective To investigate the effects of Nel-like type 1 molecule (NELL-1) on the proliferation and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHEDs). Methods SHEDs was isolated, cultured, and identified. The third generation SHEDs were used for subsequent experiments. SHEDs were divided into 4 groups, and NELL-1 was added to each group at concentrations of 0 (control group), 50, 100, and 200 ng/mL. Cell activity was measured by CCK-8 assay and crystal violet staining. The changes in osteogenic ability were detected by alkaline phosphatase activity. The expression levels of osteogenesis-related genes ALP, OCN and Runx-2 by RT-qPCR were detected. Western blot was used to detect the expression of osteogenesis-related proteins ALP, Runx-2 and Col-Ⅰ. Results SHEDs exhibited stem cell characteristics, and 50 ng/mL of NELL-1 protein had a promoting effect on SHEDs proliferation (P<0.01). Alkaline phosphatase activity assay showed that after the addition of NELL-1, the osteogenic effect of each group was better than that of the control group and 50 ng/mL of NELL-1 was the best (P<0.05). RT-qPCR and Western blot results showed that 50 ng/mL of NELL-1 significantly promoted the expression of osteoblast-related genes including ALP, OCN and Runx-2 and the protein expression of ALP, Runx-2 and Col-Ⅰ (P<0.05). Conclusion NELL-1 can promote the proliferation and osteogenic differentiation of SHEDs.

Key words: Nel-like type 1 molecule, stem cells from human exfoliated deciduous teeth, osteogenic differentiation

中图分类号:

R781

于晨, 贺龙飞, 冯瑶, 葛潇, 崔云婷, 李倜, 于淼. 神经表皮生长因子样蛋白1对人脱落乳牙牙髓干细胞增殖和骨向分化的影响[J]. 口腔医学, 2025, 45(7): 518-524.

YU Chen, HE Longfei, FENG Yao, GE Xiao, CUI Yunting, LI Ti, YU Miao. The effect of Nel-like type 1 molecule on the proliferation and osteogenic differentiation of stem cells from human exfoliated deciduous teeth[J]. Stomatology, 2025, 45(7): 518-524.

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参考文献 30

[1]	Huang GT, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources: Their biology and role in regenerative medicine[J]. J Dent Res, 2009, 88(9): 792-806. doi: 10.1177/0022034509340867 pmid: 19767575
[2]	徐丽, 布春青, 康洁, 等. MIRB标记人脱落乳牙牙髓干细胞移植修复大鼠牙槽骨缺损的实验研究[J]. 上海口腔医学, 2022, 31(6): 581-587.
[3]	da Silva AAF, Rinco UGR, Jacob RGM, et al. The effectiveness of hydroxyapatite-beta tricalcium phosphate incorporated into stem cells from human exfoliated deciduous teeth for reconstruction of rat calvarial bone defects[J]. Clin Oral Investig, 2022, 26(1): 595-608.
[4]	Hiraki T, Kunimatsu R, Nakajima K, et al. Stem cell-derived conditioned media from human exfoliated deciduous teeth promote bone regeneration[J]. Oral Dis, 2020, 26(2): 381-390. doi: 10.1111/odi.13244 pmid: 31808229
[5]	Zhang X, Zara J, Siu RK, et al. The role of NELL-1, a growth factor associated with craniosynostosis, in promoting bone regeneration[J]. J Dent Res, 2010, 89(9): 865-878. doi: 10.1177/0022034510376401 pmid: 20647499
[6]	胡诗余, 施洁珺, 胡济安. NELL-1促成骨和成软骨的作用机制及应用前景[J]. 口腔医学, 2021, 41(6): 545-550, 566.
[7]	Lee JH, Song YM, Min SK, et al. NELL-1 increased the osteogenic differentiation and mRNA expression of spheroids composed of stem cells[J]. Medicina, 2021, 57(6): 586.
[8]	Yu LY, Cen X, Xia K, et al. MicroRNA expression profiles and the potential competing endogenous RNA networks in NELL-1-induced human adipose-derived stem cell osteogenic differentiation[J]. J Cell Biochem, 2020, 121(11): 4623-4641. doi: 10.1002/jcb.29695 pmid: 32065449
[9]	Pang S, Shen J, Liu Y, et al. Proliferation and osteogenic differentiation of mesenchymal stem cells induced by a short isoform of NELL-1[J]. Stem Cells, 2015, 33(3): 904-915. doi: 10.1002/stem.1884 pmid: 25376942
[10]	Zhang N, Lu XJ, Wu SC, et al. Intrastriatal transplantation of stem cells from human exfoliated deciduous teeth reduces motor defects in Parkinsonian rats[J]. Cytotherapy, 2018, 20(5): 670-686. doi: S1465-3249(18)30407-9 pmid: 29576501
[11]	Yuniartha R, Yamaza T, Sonoda S, et al. Cholangiogenic potential of human deciduous pulp stem cell-converted hepatocyte-like cells[J]. Stem Cell Res Ther, 2021, 12(1): 57. doi: 10.1186/s13287-020-02113-8 pmid: 33436050
[12]	Kanafi MM, Rajeshwari YB, Gupta S, et al. Transplantation of islet-like cell clusters derived from human dental pulp stem cells restores normoglycemia in diabetic mice[J]. Cytotherapy, 2013, 15(10): 1228-1236. doi: 10.1016/j.jcyt.2013.05.008 pmid: 23845187
[13]	Zeng L, He H, Sun MJ, et al. Runx2 and Nell-1 in dental follicle progenitor cells regulate bone remodeling and tooth eruption[J]. Stem Cell Res Ther, 2022, 13(1): 486. doi: 10.1186/s13287-022-03140-3 pmid: 36175952
[14]	Li C, Zhang X, Zheng Z, et al. Nell-1 is a key functional modulator in osteochondrogenesis and beyond[J]. J Dent Res, 2019, 98(13): 1458-1468. doi: 10.1177/0022034519882000 pmid: 31610747
[15]	Chadipiralla K, Yochim JM, Bahuleyan B, et al. Osteogenic differentiation of stem cells derived from human periodontal ligaments and pulp of human exfoliated deciduous teeth[J]. Cell Tissue Res, 2010, 340(2): 323-333. doi: 10.1007/s00441-010-0953-0 pmid: 20309582
[16]	岳海云, 孙银雪, 毕迎春. 白藜芦醇促进人乳牙牙髓干细胞的增殖和成骨分化[J]. 中国组织工程研究, 2023, 27(19): 3011-3016.
[17]	Ning T, Shao J, Zhang X, et al. Ageing affects the proliferation and mineralization of rat dental pulp stem cells under inflammatory conditions[J]. Int Endod J, 2020, 53(1): 72-83. doi: 10.1111/iej.13205 pmid: 31419325
[18]	Ma R, Wang ML, Shi PL, et al. Effect of lipoxin A4 on the osteogenic differentiation of periodontal ligament stem cells under lipopolysaccharide-induced inflammatory conditions[J]. Eur J Oral Sci, 2023, 131(3): e12932.
[19]	Vallés G, Bensiamar F, Maestro-Paramio L, et al. Influence of inflammatory conditions provided by macrophages on osteogenic ability of mesenchymal stem cells[J]. Stem Cell Res Ther, 2020, 11(1): 57. doi: 10.1186/s13287-020-1578-1 pmid: 32054534
[20]	Chhabra R, Rao SM, Kumar BM, et al. Characterization of stem cells from human exfoliated deciduous anterior teeth with varying levels of root resorption[J]. J Clin Pediatr Dent, 2021, 45(2): 104-111.
[21]	Zhang XL, Péault B, Chen WW, et al. The Nell-1 growth factor stimulates bone formation by purified human perivascular cells[J]. Tissue Eng Part A, 2011, 17(19/20): 2497-2509.
[22]	Vimalraj S. Alkaline phosphatase: Structure, expression and its function in bone mineralization[J]. Gene, 2020, 754: 144855.
[23]	程德斌, 张昭, 付军, 等. 不同孔径3D打印三周期极小曲面基元支架调控成骨细胞生物学行为的研究[J]. 实用骨科杂志, 2023, 29(11): 994-1000.
[24]	Liu C, Xiong H, Chen K, et al. Long-term exposure to pro-inflammatory cytokines inhibits the osteogenic/dentinogenic differentia-tion of stem cells from the apical papilla[J]. Int Endod J, 2016, 49(10): 950-959. doi: 10.1111/iej.12551 pmid: 26383494
[25]	徐静, 吕慧欣, 王一涵, 等. Nell-1蛋白通过Wnt/β-catenin通路调控人上颌窦黏膜来源间充质干细胞成骨分化[J]. 口腔医学研究, 2023, 39(3): 227-235. doi: 10.13701/j.cnki.kqyxyj.2023.03.009
[26]	李彦秋, 李祖兵. 结缔组织生长因子对NELL-1诱导的MC3T3-E1细胞成骨分化的影响[J]. 口腔生物医学, 2023, 14(3): 169-174.
[27]	韩琦, 王效英. 牙髓血管再生的相关研究进展[J]. 口腔医学, 2019, 39(4): 371-375.
[28]	Pakvasa M, Alverdy A, Mostafa S, et al. Neural EGF-like protein 1 (NELL-1): Signaling crosstalk in mesenchymal stem cells and applications in regenerative medicine[J]. Genes Dis, 2017, 4(3): 127-137. doi: 10.1016/j.gendis.2017.07.006 pmid: 29276737
[29]	Shen MJ, Wang GG, Wang YZ, et al. Nell-1 enhances osteogenic differentiation of pre-osteoblasts on titanium surfaces via the MAPK-ERK signaling pathway[J]. Cell Physiol Biochem, 2018, 50(4): 1522-1534.
[30]	James AW, Shen J, Tsuei R, et al. NELL-1 induces Sca-1⁺ mesenchymal progenitor cell expansion in models of bone maintenance and repair[J]. JCI Insight, 2017, 2(12): e92573.

基因名称	引物序列(5'→3')
ALP	F:GGCGGTGAACGAGAGAATGT R:GGACGTAGTTCTGCTCGTGG
Runx-2	F:GGAGTGGACGAGGCAAGAGT R:AGGCGGTCAGAGAACAAACT
OCN	F:TCACACTCCTCGCCCTATTG R:CTCTTCACTACCTCGCTGCC
GAPDH	F:GCACCGTCAAGGCTGAGAAC R:TGGTGAAGACGCCAGTGGA