Nuclear factor I-C regulates differentiation of human stem cells from apical papilla

doi:10.12307/2025.649

Abstract

Abstract: BACKGROUND: Overexpression of the nuclear factor I-C gene in vitro promotes the differentiation of human stem cells from apical papilla, as does the activation of the Wnt/β-catenin signaling pathway. Moreover, nuclear factor I-C regulates the Wnt/β-catenin pathway in mesenchymal stem cells. However, whether nuclear factor I-C can affect cell differentiation by activating the Wnt/β-catenin pathway in human stem cells from apical papilla has not been reported.
OBJECTIVE: To investigate the role of nuclear factor I-C in the Wnt/β-catenin signaling pathway in regulating the differentiation of human stem cells from apical papilla.
METHODS: Human stem cells from apical papilla were cultured by the slide-covered tissue block method and lentiviral transfection overexpressing the nuclear factor I-C gene. (1) A control group, an empty viral vector group, and an overexpressed nuclear factor I-C gene group were set up. The expression of β-Catenin, LRP5, and TCF7L2 was detected by Western blotting. (2) The control group, empty viral vector group, overexpressed nuclear factor I-C gene group, and overexpressed nuclear factor I-C gene+DKK-1 (Wnt pathway inhibitor) group were set up. Alkaline phosphatase staining and activity quantification were performed after 7 days of osteogenic induction. qPCR and Western blotting were performed to detect the expression of Runt-related transcription factor 2, dentin salivary phosphoprotein, osteocalcin mRNA, and protein after 14 days of osteogenic induction. Alizarin Red staining was used to observe the formation of mineralized nodules.
RESULTS AND CONCLUSION: (1) Compared with the control and empty viral vector groups, the expression of Wnt/β-Catenin pathway-related proteins β-Catenin, LRP5, and TCF7L2 in human apical dentin papilla stem cells was significantly increased in the overexpressed nuclear factor I-C gene group (P < 0.01). (2) Compared with the control and empty viral vector groups, the expression of alkaline phosphatase and osteocalcin in human apical dentin papilla stem cells was significantly increased (P < 0.01); the expression levels of Runt-related transcription factor 2, dentin salivary phosphoprotein, osteocalcin mRNA and protein were significantly higher (P < 0.01), and the number of mineralized nodules was significantly increased (P < 0.01) in the overexpressed nuclear factor I-C gene group. (3) Compared with the overexpressed nuclear factor I-C gene group, the alkaline phosphatase activity and the expression of Runt-related transcription factor 2, dentin salivary phosphoprotein, osteocalcin mRNA and protein expression levels were significantly down-regulated (P < 0.05), and the number of mineralized nodules was significantly reduced (P < 0.05) in human stem cells from apical papilla of the overexpressed nuclear factor I-C gene+DKK-1 group. The results show that nuclear factor I-C can activate the Wnt/β-catenin signaling pathway in human stem cells from apical papilla and mediate the osteogenic/odontogenic differentiation of human stem cells from apical papilla.

Key words: 人根尖牙乳头干细胞, 核因子I-C, Wnt, 信号通路, 分化, 成骨, DKK-1, 工程化干细胞

CLC Number:

Wu Yue, Zhu Yongna, Ge Xiang, Liu Fan, He Zeyu, Liu Xi. Nuclear factor I-C regulates differentiation of human stem cells from apical papilla[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(31): 6667-6673.

Figures/Tables 2

2.1 人根尖牙乳头干细胞的形态组织取材自新鲜拔除的牙根尚未发育完全的恒牙(图1A)，玻片覆盖组织块法培养原代根尖牙乳头干细胞，培养五六天后发现组织块周围有不规则多角形细胞爬出(图1B)，培养9-10 d后组织块周围大量细胞聚集(图1C)，细胞呈长梭形，以组织块为中心放射状生长。细胞融合度在70%-80%时，胰酶消化传代，扩大培养(图1D)。 2.2 人根尖牙乳头干细胞表面特异抗原鉴定通过流式细胞仪对人根尖牙乳头干细胞表面抗原进行检测，结果显示细胞高表达间充质干细胞表面特异性抗原，CD44、CD90、CD105表达阳性率均> 90%，低表达造血干细胞表面抗原，CD45表达阳性率< 5%(图2)。 2.3 人根尖牙乳头干细胞多向分化潜能鉴定人根尖牙乳头干细胞成骨诱导3周后，镜下可见细胞间紧密相连成片。茜素红染色显示，细胞之间出现了暗红色的矿化结节(图3A)。人根尖牙乳头干细胞成脂诱导3周后，油红O染色显示，在细胞质中可以看到暗红色的脂滴形成(图3B)，上述结果表明该细胞具有成骨、成脂分化能力。 2.4 建立过表达核因子I-C基因的人根尖牙乳头干细胞系转染72 h后，Western blot检测结果显示，过表达核因子I-C组的核因子I-C蛋白表达量显著高于其他两组(图4)，差异有显著性意义(P < 0.01)，对照组与空载体组的核因子I-C蛋白表达量无统计学差异，说明过表达核因子I-C慢病毒对人根尖牙乳头干细胞感染有效，而感染空病毒载体并不会影响细胞核因子I-C基因的表达。 2.5 β-Catenin、LRP5、TCF7L2蛋白表达水平过表达核因子I-C组Wnt/β-catenin信号通路相关β-catenin、LRP5、TCF7L2蛋白表达水平显著高于对照组(P < 0.01)，对照组与空载体组的β-catenin、LRP5、TCF7L2蛋白表达水平无统计学差异(图5)。上述结果表明，核因子I-C可以促进人根尖牙乳头干细胞中Wnt/β-catenin信号通路的激活，空病毒载体对通路的激活无实质性影响。 2.6 NFIC/Wnt/β-Catenin信号对人根尖牙乳头干细胞分化的影响 2.6.1 NFIC/Wnt/β-Catenin信号对人根尖牙乳头干细胞早期矿化能力的影响各组人根尖牙乳头干细胞成骨诱导7 d后进行碱性磷酸酶染色，可见过表达核因子I-C组较空白对照组、空载体组、过表达核因子I-C+DKK-1组染色深(图6A)。过表达核因子I-C组碱性磷酸酶活性高于其他3组(P < 0.01)(图6B)。 "

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