转录因子TBX1调控人牙髓间充质干细胞成骨分化的作用研究

摘要/Abstract

摘要：

目的探讨T-box转录因子1(T-box transcription factor 1,TBX1)对人牙髓间充质干细胞(human dental pulp stem cells,hDPSCs)成骨分化的调控作用及其潜在机制。方法体外使用慢病毒转染技术构建TBX1敲低的hDPSCs模型,通过碱性磷酸酶(alkaline phosphatase,ALP)染色检测成骨诱导早期细胞分化活性,通过茜素红S(alizarin red S,ARS)染色观察成骨晚期矿化结节的形成情况;并结合Western blot及RT-qPCR检测关键成骨分化标志物Ⅰ型胶原(collagen type Ⅰ,COL1)、成骨细胞特异性转录因子(osterix,OSX)及Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)在蛋白及mRNA水平的表达变化。收集成骨诱导第7天条件培养基进行人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)体外成管实验,同时采用RT-qPCR检测hDPSCs血管内皮生长因子A(vascular endothelial growth factor A,VEGFA)、血管生成素-1(angiopoietin-1,ANG1)、血小板衍生生长因子B(platelet-derived growth factor subunit B,PDGFB)的mRNA表达水平。体内采用裸鼠皮下异位成骨模型,随机分为对照组(consh)与TBX1敲低组(TBX1sh),分别于背侧皮下植入转染consh或TBX1sh的hDPSCs与羟基磷灰石/磷酸三钙(HA/TCP)材料构建的复合材料,通过HE染色比较对照组与TBX1敲低组新生骨的形成差异,通过免疫组化染色检测成骨分化相关蛋白RUNX2、骨钙素(osteocalcin, OCN)、骨涎蛋白(bone sialoprotein,BSP)及血小板-内皮细胞黏附分子(platelet endothelial cell adhesion molecule,CD31)表达。结果体外实验发现,敲低TBX1显著增强hDPSCs的ALP活性、矿化结节形成及成骨分化相关标志物(COL1、OSX、RUNX2)表达(P<0.05),VEGFA、ANG1、PDGFB的mRNA水平显著上调(P<0.05),其条件培养基亦显著促进HUVECs管状结构形成(P<0.05)。体内实验证实,TBX1敲低组裸鼠皮下新生骨组织形成增加(P<0.05),成骨分化相关蛋白表达上调(P<0.05),CD31免疫组化染色显示微血管密度及积分光密度值均显著高于对照组(P<0.05)。结论 TBX1是hDPSCs成骨分化的负向调控因子,敲低TBX1可有效促进hDPSCs成骨分化,其机制可能与增强细胞促血管生成能力、改善局部微环境有关。

关键词: TBX1, 人牙髓间充质干细胞, 成骨分化, 骨再生

Abstract:

Objective To investigate the regulatory effect of T-box transcription factor 1 (TBX1) on the osteogenic differentiation capacity of human dental pulp stem cells (hDPSCs) and its underlying mechanism. Methods In vitro, a TBX1-knockdown hDPSCs model was constructed using lentiviral transfection. Alkaline phosphatase (ALP) staining was performed to assess early osteogenic differentiation, and alizarin red S (ARS) staining was used to observe mineralized nodule formation at the late stage. The expression levels of osteogenic markers including collagen type Ⅰ (COL1), osterix (OSX), and Runt-related transcription factor 2 (RUNX2) were detected by Western blot and RT-qPCR. Conditioned medium collected on day 7 of osteogenic induction was used for tube formation assay with human umbilical vein endothelial cells (HUVECs), and the mRNA expression levels of vascular endothelial growth factor A (VEGFA), angiopoietin-1 (ANG1), and platelet-derived growth factor subunit B (PDGFB) were detected by RT-qPCR. In vivo, an ectopic osteogenesis model was established in nude mice by subcutaneous implantation on the dorsum. The mice were randomly divided into control group (consh) and TBX1 knockdown group (TBX1sh), which received composites of HA/TCP scaffolds seeded with hDPSCs transfected with consh or TBX1sh, respectively. HE staining was performed to compare the differences in new bone formation between the control and TBX1 knockdown groups. Immunohistochemi-cal staining was conducted to detect the expression of osteogenesis-related proteins, including RUNX2, osteocalcin(OCN), bone sialoprotein(BSP), and platelet endothelial cell adhesion molecule (CD31). Results In vitro experiments showed that TBX1 knockdown significantly enhanced ALP activity, mineralized nodule formation, and the expression of osteogenic differentiation markers (COL1, OSX, RUNX2) in hDPSCs(P<0.05). Additionally, the mRNA levels of VEGFA, ANG1, and PDGFB were significantly upregulated (P<0.05), and the conditioned medium from TBX1-knockdown hDPSCs markedly promoted the formation of tubular structures in HUVECs (P<0.05). In vivo results demonstrated increased new bone formation and upregulated expression of osteogenic-related proteins in the TBX1-knockdown group (P<0.05). CD31 immunohistochemical staining showed that both microvessel density and integrated optical density were significantly higher in the TBX1-knockdown group compared to the control group (P<0.05). Conclusion TBX1 is a negative regulator of osteogenic differentiation in hDPSCs. Knockdown of TBX1 effectively promotes the osteogenic differentiation of hDPSCs, and the underlying mechanism may involve enhanced pro-angiogenic capacity and improved local microenvironment.

Key words: TBX1, human dental pulp stem cells (hDPSCs), osteogenic differentiation, bone regeneration

中图分类号:

R781.3

王明熙, 高姗, 李国情, 汤春波. 转录因子TBX1调控人牙髓间充质干细胞成骨分化的作用研究[J]. 口腔医学, 2026, 46(6): 427-434.

WANG Mingxi, GAO Shan, LI Guoqing, TANG Chunbo. Regulatory effect of transcription factor TBX1 on osteogenic differentiation of human dental pulp stem cells[J]. Stomatology, 2026, 46(6): 427-434.

图/表 11

表1

图1

图2

图3

图4

图5

图6

图7

图8

图9

图10

参考文献 32

[1]	Raymond Y, Pastorino D, Ginebreda I, et al. Computed tomography and histological evaluation of xenogenic and biomimetic bone grafts in three-wall alveolar defects in minipigs[J]. Clin Oral Investig, 2021, 25(12): 6695-6706. doi: 10.1007/s00784-021-03956-y
[2]	Zhao J, Zhou YH, Zhao YQ, et al. Oral cavity-derived stem cells and preclinical models of jaw-bone defects for bone tissue engineering[J]. Stem Cell Res Ther, 2023, 14(1): 39. doi: 10.1186/s13287-023-03265-z pmid: 36927449
[3]	Apatzidou DA, Iliopoulos JM, Konstantinidis A, et al. Inflammatory and bone remodelling related biomarkers following periodontal transplantation of the tissue engineered bio complex[J]. Clin Oral Investig, 2024, 28(7): 361. doi: 10.1007/s00784-024-05754-8
[4]	Wang WQ, Yang HQ, Fan ZP, et al. NQO1 promotes osteogenesis and suppresses angiogenesis in DPSCs via MAPK pathway modulation[J]. Stem Cell Res Ther, 2024, 15(1): 306. doi: 10.1186/s13287-024-03929-4
[5]	Zheng XY, Wang J, Zhou H, et al. Dental pulp stem cells alleviate Schwann cell pyroptosis via mitochondrial transfer to enhance facial nerve regeneration[J]. Bioact Mater, 2025, 47: 313-326.
[6]	袁忠治, 胡伟平. 牙髓干细胞对中枢神经系统疾病的治疗新进展[J]. 口腔医学, 2018, 38(5): 471-475.
[7]	Liu Y, Liu YT, Hu JC, et al. Impact of allogeneic dental pulp stem cell injection on tissue regeneration in periodontitis: A multicenter randomized clinical trial[J]. Signal Transduct Target Ther, 2025, 10(1): 239.
[8]	Yu JH, He HX, Tang CB, et al. Differentiation potential of STRO-1⁺ dental pulp stem cells changes during cell passaging[J]. BMC Cell Biol, 2010, 11: 32. doi: 10.1186/1471-2121-11-32
[9]	Tatullo M, Marrelli M, Shakesheff KM, et al. Dental pulp stem cells: Function, isolation and applications in regenerative medicine[J]. J Tissue Eng Regen Med, 2015, 9(11): 1205-1216. doi: 10.1002/term.1899 pmid: 24850632
[10]	Cen HM, Luo H, Luo B, et al. TBX1 regulates myogenic differentiation by activating the TGFβ-Smad2/3 pathway in myoblasts[J]. Exp Biol Med, 2023, 248(1): 61-69. doi: 10.1177/15353702221112087
[11]	Eom TY, Schmitt JE, Li YR, et al. Tbx1 haploinsufficiency leads to local skull deformity, paraflocculus and flocculus dysplasia, and motor-learning deficit in 22q11.2 deletion syndrome[J]. Nat Commun, 2024, 15(1): 10510. doi: 10.1038/s41467-024-54837-3
[12]	Fulcoli FG, Franzese M, Liu XY, et al. Rebalancing gene haploinsufficiency in vivo by targeting chromatin[J]. Nat Commun, 2016, 7: 11688. doi: 10.1038/ncomms11688
[13]	Dastjerdi A, Robson L, Walker R, et al. Tbx1 regulation of myogenic differentiation in the limb and cranial mesoderm[J]. Dev Dyn, 2007, 236(2): 353-363. doi: 10.1002/dvdy.v236:2
[14]	Kelly RG, Jerome-Majewska LA, Papaioannou VE. The del22q11.2 candidate gene Tbx1 regulates branchiomeric myogenesis[J]. Hum Mol Genet, 2004, 13(22): 2829-2840. pmid: 15385444
[15]	Khotib J, Marhaeny HD, Miatmoko A, et al. Differentiation of osteoblasts: The links between essential transcription factors[J]. J Biomol Struct Dyn, 2023, 41(19): 10257-10276. doi: 10.1080/07391102.2022.2148749
[16]	Hilkens P, Gervois P, Fanton Y, et al. Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells[J]. Cell Tissue Res, 2013, 353(1): 65-78. doi: 10.1007/s00441-013-1630-x pmid: 23715720
[17]	Wang W, Zhu YR, Liu Y, et al. 3D bioprinting of DPSCs with GelMA hydrogel of various concentrations for bone regeneration[J]. Tissue Cell, 2024, 88: 102418. doi: 10.1016/j.tice.2024.102418
[18]	Mattei V, Martellucci S, Pulcini F, et al. Regenerative potential of DPSCs and revascularization: Direct, paracrine or autocrine effect?[J]. Stem Cell Rev Rep, 2021, 17(5): 1635-1646. doi: 10.1007/s12015-021-10162-6 pmid: 33829353
[19]	Leyendecker Junior A, Gomes Pinheiro CC, Lazzaretti Fernandes T, et al. The use of human dental pulp stem cells for in vivo bone tissue engineering: A systematic review[J]. J Tissue Eng, 2018, 9: 2041731417752766.
[20]	Liu FQ, Wu QQ, Liu QW, et al. Dental pulp stem cells-derived cannabidiol-treated organoid-like microspheroids show robust osteogenic potential via upregulation of WNT6[J]. Commun Biol, 2024, 7(1): 972. doi: 10.1038/s42003-024-06655-y
[21]	Kornsuthisopon C, Nowwarote N, Srisuwan T, et al. Regulatory pathways governing odonto/osteogenic differentiation in dental pulp stem cells[J]. Int Endod J, 2026, 59(3): 372-395. doi: 10.1111/iej.v59.3
[22]	Aval SF, Lotfi H, Sheervalilou R, et al. Tuning of major signaling networks (TGF-β, Wnt, Notch and Hedgehog) by miRNAs in human stem cells commitment to different lineages: Possible clinical application[J]. Biomed Pharmacother, 2017, 91: 849-860. doi: S0753-3322(17)31198-8 pmid: 28501774
[23]	Jerome LA, Papaioannou VE. DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1[J]. Nat Genet, 2001, 27(3): 286-291. doi: 10.1038/85845 pmid: 11242110
[24]	Komori T. Whole aspect of Runx2 functions in skeletal development[J]. Int J Mol Sci, 2022, 23(10): 5776. doi: 10.3390/ijms23105776
[25]	Funato N, Srivastava D, Shibata S, et al. TBX1 regulates chondrocyte maturation in the spheno-occipital synchondrosis[J]. J Dent Res, 2020, 99(10): 1182-1191. doi: 10.1177/0022034520925080 pmid: 32442036
[26]	Papangeli I, Scambler P. The 22q11 deletion: DiGeorge and velocardiofacial syndromes and the role of TBX1[J]. Wiley Interdiscip Rev Dev Biol, 2013, 2(3): 393-403. doi: 10.1002/wdev.v2.3
[27]	Komori T. Regulation of skeletal development and maintenance by Runx2 and Sp7[J]. Int J Mol Sci, 2024, 25(18): 10102. doi: 10.3390/ijms251810102
[28]	Shen JJ, Sun Y, Liu XZ, et al. EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling[J]. Stem Cell Res Ther, 2021, 12(1): 415. doi: 10.1186/s13287-021-02487-3
[29]	Cioffi S, Martucciello S, Fulcoli FG, et al. Tbx1 regulates brain vascularization[J]. Hum Mol Genet, 2014, 23(1): 78-89. doi: 10.1093/hmg/ddt400 pmid: 23945394
[30]	Lungu CN, Mehedinti MC. Molecular motifs in vascular morphogenesis: Vascular endothelial growth factor A (VEGFA) as the leading promoter of angiogenesis[J]. Int J Mol Sci, 2023, 24(15): 12169. doi: 10.3390/ijms241512169
[31]	Scott MA, Levi B, Askarinam A, et al. Brief review of models of ectopic bone formation[J]. Stem Cells Dev, 2012, 21(5): 655-667. doi: 10.1089/scd.2011.0517 pmid: 22085228
[32]	Burckhardt K, Jung O, Stošić MR, et al. Comparative evaluation of biocompatibility data from the subcutaneous and the calvaria implantation model of a novel bone substitute block[J]. In Vivo, 2025, 39(6): 3116-3127. doi: 10.21873/invivo.14113

基因	引物序列(5'-3')
TBX1	F:GCTGTGGGACGAGTTCAATC R:ACGTGGGGAACATTCGTCT
COL1	F:GCTACTACCGGGCTGATGAT R:ATGTTCTCGATCTGCTGGCT
OSX	F:CCCACCTCAGGCTATGCTAA R:CATGGATGCCTGCCTTGTAC
RUNX2	F:TGAACTCTGCACCAAGTCCT R:GGGTGGTAGAGTGGATGGAC
GAPDH	F:TCGTGGAAGGACTCATGACC R:ATGATGTTCTGGAGAGCCCC
VEGFA	F:GTGAGGTTTGATCCGCATGAT R:GACCCTGGCTTTACTGCTGTA
ANG1	F:CTACCAACAACAACAGCATCC R:CTCCCTTTAGCAAAACACCTTC
PDGFB	F:ATGAAATGCTGAGCGACCAC G:TCCCTCGAGATGAGCTTTCC