Chir99021调控Wnt/β-catenin 信号通路抑制大鼠牙髓干细胞成骨诱导分化的机制

doi:10.16098/j.issn.0529-1356.2024.01.010

摘要/Abstract

摘要：

目的探讨Chir99021对大鼠牙髓干细胞（DPSCs）成骨诱导分化的影响及机制。方法原代分离大鼠DPSCs，利用荧光免疫法进行验证。设置不同浓度的Chir99021，通过CCK-8检测细胞增殖情况挑选最适浓度。利用茜素红染色验证细胞成骨诱导分化。最后通过Real-time PCR、Western blotting检测成骨诱导分化相关基因与蛋白无翅型MMTV整合位点家族成员1（Wnt1）、无翅型MMTV 整合位点家族成员3A（Wnt3a）、β-连环蛋白（β-catenin）、轴抑制蛋白2抗体（Axin2）、骨钙素（OCN）、牙本质基质蛋白1（DMP1）的mRNA和蛋白表达情况。结果牙本质涎磷蛋白（DSPP）和干细胞标记物波形蛋白（vimentin）阳性表达，说明大鼠DPSCs分离成功。大鼠DPSCs成骨诱导后钙沉积物显著上升，加入糖原合成酶激酶-3β（GSK-3β）的小分子抑制剂Chir99021后钙沉积物明显减少。大鼠DPSCs成骨诱导分化后Wnt1、Wnt3a、β-catenin、Axin2、OCN和DMP1表达明显上升，而加入Chir99021后Wnt1、Wnt3a、β-catenin、Axin2、OCN和DMP1蛋白表达均显著下降。结论 Chir99021对诱导7 d后的大鼠DPSCs成骨诱导分化有一定的抑制作用。

关键词: 牙髓干细胞, 成骨诱导分化, Chir99021, 实时定量聚合酶链反应, 大鼠

Abstract:

Objective To explore the effect and mechanism of Chir99021 on osteogenic differentiation of rat dental pulp stem cells. Methods Primary rat dental pulp stem cells were isolated from rat dental pulp and verified by fluorescence immunoassay. Different concentrations of Chir99021 were set, and the cell proliferation was detected by CCK-8 to select the optimal concentration. Osteogenic differentiation was detected by alizarin red staining. The expression of osteogenic differentiation related genes and proteins recombinant wingless type MMTV integration site famity member 1(Wnt1), Wnt3a and Wnt3a β-expression of catenin, axis inhibition protein 2(Axin 2), dentin sialophosphoprotein(OCN) and dentin matrix acidic phosphoprotein 1(DMP1) was detected by Real-time PCR and Western blotting. Results The positive expression of dentin sialophosphoprotein(DSPP) and vimentin indicated that rat dental pulp stem cells were successfully isolated. After osteogenic induction of rat dental pulp stem cells, calcium deposits significantly increased with the addition of glycogen synthase kinase-3β(GSK-3β) inhibitor Chir99021, calcium deposits were significanted reduced. After osteogenic differentiation of rat dental pulp stem cells, the expression of Wnt1, Wnt3a, β-catenin, Axin2, OCN and DMP1 increased, while the expression of Wnt1, Axin2, OCN and DMP1 decreased with the addition of Chir99021. Conclusion Chir99021 can inhibit the osteogenic differentiation of rat dental pulp stem cells after 7 days of induction.

Key words: Dental pulp stem cell, Osteogenic differentiation, Chir99021, Real-time PCR, Rat

中图分类号:

谢惠兰方芳林毅. Chir99021调控Wnt/β-catenin 信号通路抑制大鼠牙髓干细胞成骨诱导分化的机制[J]. 解剖学报, 2024, 55(1): 67-72.

XIE Hui-lan FANG Fang LIN Yi. Mechanism of Chir99021 regulating Wnt/β-catenin signaling pathway inhibiting osteogenic differentiation of rat dental pulp stem cells[J]. Acta Anatomica Sinica, 2024, 55(1): 67-72.

参考文献

［1］Ding G, Liu Y, Wang SL. Research status of dental pulpstem cells［J］. Journal of Capital Medical University, 2008,5:660-663. (in Chinese)
丁刚,刘怡,王松灵.牙髓干细胞研究现状［J］.首都医科大学学报,2008,5:660-663.
［2］Uribe-Etxebarria V, Pineda JR, García-Gallastegi P, et al. Notch and Wnt signaling modulation to enhance DPSC stemness and therapeutic potential［J］. Int J Mol Sci, 2023, 24(8):7389.
［3］Damrongsri D, Nowwarote N, Sonpoung O, et al. Differential expression of Notch related genes in dental pulp stem cells and stem cells isolated from apical papilla［J］. J Oral Biol Craniofac Res, 2021, 11(3):379-385.
［4］Feng G, Zheng K, Song D, et al. SIRT1 was involved in TNF-α-promoted osteogenic differentiation of human DPSCs through Wnt/β-catenin signal［J］. In Vitro Cell Dev Biol Anim, 2016,52(10):1001-1011.
［5］Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo［J］. Proc Natl Acad Sci USA, 2000,97(25):13625-13630.
［6］Xi J, Liu Y, Liu H, et al. Specification of midbrain dopamine neurons from primate pluripotent stem cells［J］. Stem Cells,2012,30(8):1655-1663.
［7］Kolios G, Moodley Y. Introduction to stem cells and regenerative medicine［J］. Respiration, 2013, 85(1):3-10.

［8］Zhan XW, Yang L, Zheng MR, et al. Research progress in the regulation of lipogenic balance of bone marrow mesenchymal stem cells ［J］. Acta Antica Sinica,2019,50(3):400-404. (in Chinese)

占秀文,杨磊,郑美蓉,等.骨髓间充质干细胞成脂成骨平衡调控研究进展［J］.解剖学报,2019,50(3):400-404.
［9］Mortada I, Mortada R, Al Bazzal M. Dental pulp stem cells and neurogenesis［J］. Adv Exp Med Biol,2018,1083: 63-75.
［10］Anitua E, Troya M, Zalduendo M. Progress in the use of dental pulp stem cells in regenerative medicine［J］. Cytotherapy,2018,20(4):479-498.
［11］Li WL, Zhang GY, Sun PX. Chemical biology in stem cell research: a new interdisciplinary frontier ［J］. Journal of Second Military Medical University, 2017, 38(2): 133-141. (in Chinese)
李文林,张冠宇,孙平新.干细胞研究中的化学生物学:学科交叉的新前沿［J］.第二军医大学学报,2017,38(2):133-141.
［12］von Wangenheim KH, Peterson HP. The role of cell differentiation in controlling cell multiplication and cancer［J］. J Cancer Res Clin Oncol,2008,134(7):725-741.
［13］Hu H, Zhao P, Liu J, et al. Lanthanum phosphate/chitosan scaffolds enhance cytocompatibility and osteogenic efficiency via the Wnt/β-catenin pathway［J］. J Nanobiotechnology,2018,16(1):98.
［14］Xu YX, Wu CL, Wu Y, et al. Epimedium-derived flavonoids modulate the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats via Wnt/β-catenin signal pathway activation［J］. Chin J Integr Med, 2012,18(12):909-917.
［15］Tóth F, T?zsér J, Hegedüs C. Effect of inducible BMP-7 expression on the osteogenic differentiation of human dental pulp stem cells［J］. Int J Mol Sci, 2021, 22(12):6182.
［16］He QJ, Zhang NN, Yin YCh, et al. Wnt/β-catenin signaling pathway promotes differentiation of mouse embryonic stem cells into insulin-secreting cells ［J］. Journal of Anhui Medical University, 2019, 54(12): 1841-1848. (in Chinese)
何巧娟,张娜娜,殷应传,等.Wnt/β-catenin信号通路促进小鼠胚胎干细胞向胰岛素分泌细胞分化［J］.安徽医科大学学报,2019,54(12):1841-1848.
［17］Cai M, Li J, Yue R, et al. Glycosylation of DMP1 maintains cranial sutures in mice［J］. J Oral Rehabil, 2020, 47（Suppl 1）:19-28.
［18］Padovano JD, Ramachandran A, Bahmanyar S, et al. Bone-specific overexpression of DMP1 influences osteogenic gene expression during endochondral and intramembranous ossification［J］. Connect Tissue Res,2014,55(Suppl 1):121-124.
［19］Zhao Q, Wang ZX, He JT. Effects of β-ecdysterone on proliferation, differentiation and apoptosis of rat osteoblasts induced by high glucose ［J］. Chinese Journal of Clinical Pharmacology, 2021, 37(4): 443-446. (in Chinese)
赵强,王字兴,何江涛.β-蜕皮甾酮对高糖诱导的大鼠成骨细胞增殖、分化和凋亡的影响［J］.中国临床药理学杂志,2021,37(4):443-446.

［20］Wei J, Karsenty G. An overview of the metabolic functions of osteocalcin［J］. Rev Endocr Metab Disord, 2015,16(2):93-98.

［21］Sun T, Annunziato S, Bergling S, et al. ZNRF3 and RNF43 cooperate to safeguard metabolic liver zonation and hepatocyte proliferation［J］. Cell Stem Cell, 2021, 28(10):1822-1837.

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