口腔疾病防治 ›› 2022, Vol. 30 ›› Issue (3): 169-177.DOI: 10.12016/j.issn.2096-1456.2022.03.003

• 基础研究 • 上一篇    下一篇

CCDC134调控人牙髓干细胞成骨分化

徐万田(), 董文睿, 朱文胤   

  1. 南京大学医学院附属口腔医院,南京市口腔医院第三门诊部,江苏 南京(210008)
  • 收稿日期:2021-07-26 修回日期:2021-09-05 出版日期:2022-03-20 发布日期:2021-12-09
  • 通讯作者: 徐万田
  • 基金资助:
    江苏省自然科学基金项目(BK20200150)

CCDC134 regulates the osteogenic differentiation of human dental pulp stem cells

XU Wantian(), DONG Wenrui, ZHU Wenyin   

  1. Department of the Third Outpatient, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing 210008, China
  • Received:2021-07-26 Revised:2021-09-05 Online:2022-03-20 Published:2021-12-09
  • Contact: XU Wantian
  • Supported by:
    Natural Science Foundation of Jiangsu Province(BK20200150)

摘要:

目的 探讨CCDC134(coiled-coil domain containing 134)对人牙髓干细胞(human dental pulp stem cells,hDPSCs)成骨分化功能的调控作用。方法 从牙髓组织中,分离培养hDPSCs,并分别以NC-CCDC134、shCCDC134、CCDC134慢病毒转染hDPSCs,分为空白对照组、阴性对照组、CCDC134下调组(shCCDC134)、CCDC134过表达组(CCDC134)。流式细胞术检测hDPSCs表面标志物Stro-1、CD105、CD34、CD45;甲苯胺蓝染色检测克隆形成;碱性磷酸酶(alkaline phosphatase,ALP)染色检测ALP表达;茜素红染色检测矿化结节形成;油红染色检测成脂能力;qPCR检测CCDC134、Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)、骨钙素(osteocalcin,OCN)、骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)、Smad家族成员1(mothers against decapentaplegic homolog 1,SMAD1)的mRNA水平表达;蛋白印迹法检测CCDC134、RUNX2、OCN、BMP-2、SMAD1蛋白表达水平。进一步以BMP-2信号激活剂(BMP-2)和抑制剂(Dorsomorphin)分别干预CCDC134下调及上调的hDPSCs(分组为:shCCDC134、shCCDC134+BMP-2、CCDC134、CCDC134+Dorsomorphin),细胞聚合体移植于裸鼠皮下2个月,HE染色法检测新骨形成。结果 hDPSCs高表达间充质干细胞表面标志物,低表达造血干细胞表面标志物。与空白对照组相比,成骨诱导的hDPSCs中CCDC134的表达升高;与阴性对照组相比,shCCDC134组CCDC134的表达降低,CCDC134组的表达升高(P < 0.05);shCCDC134组的矿化结节减少、成骨相关基因和蛋白表达降低(P < 0.05),CCDC134组的指标升高(P < 0.05);shCCDC134组的BMP-2/SMAD1信号通路的相关表达降低,CCDC134组表达升高(P < 0.05)。与shCCDC134组相比,shCCDC134+BMP-2组成骨相关基因和蛋白表达 升高、裸鼠皮下新骨形成增加(P < 0.05),与CCDC134组相比,CCDC134+Dorsomorphin组以上指标降低(P < 0.05)。结论 CCDC134通过调控BMP-2/SMAD1信号通路促进hDPSCs成骨分化。

关键词: 牙髓干细胞, CCDC134, 成骨分化, 组织工程, 骨形成蛋白-2, 重组人Smad家族成员1, Runt相关转录因子2, 骨钙素

Abstract:

Objective To study the regulatory effect of coiled-coil domain containing 134 (CCDC134) on the osteogenic differentiation of human dental pulp stem cells (hDPSCs).Methods HDPSCs were isolated and cultured from dental pulp tissue and transfected with NC-CCDC134, shCCDC134 and CCDC134 lentiviruses. They were divided into the control group, negative control group, CCDC134 downregulation (shCCDC134) group and CCDC134 overexpression (CCDC134) group. Surface markers of hDPSCs (Stro-1, CD105, CD34, CD45) were detected by flow cytometry; colony formation was analyzed by toluidine blue staining; ALP expression was estimated by ALP staining; mineralized nodule formation was evaluated by alizarin red staining; lipid droplet formation was examined by oil red staining; and gene and protein expression of CCDC134, Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), bone morphogenetic protein-2 (BMP-2), and mothers against decapentaplegic homolog 1 (SMAD1) was detected by qPCR and western blot, respectively. Further, a BMP-2 activator (BMP-2) and inhibitor (Dorsomorphin) were used to down-regulate and up-regulate CCDC134, respectively (shCCDC134, shCCDC134+BMP-2, CCDC134, CCDC134+Dorsomorphin), in hDPSCs. The hDPSC aggregates were subcutaneously transplanted into nude mice for 2 months, and new bone formation was detected by H&E staining. The BMP-2/SMAD1 signaling in each group was detected by qPCR.Results hDPSCs showed high expression of mesenchymal markers and low expression of hematopoietic markers. Compared with the control group, the expression of CCDC134 was increased in the osteogenic-induced hDPSCs (P < 0.05). Compared with the negative control group, the expression of CCDC134 was decreased in the shCCDC134 group, whereas it was increased in the CCDC134 group (P < 0.05). The mineralized nodules, osteogenic genes and proteins in the shCCDC134 group were decreased (P < 0.05), while they were increased in the CCDC134 group (P < 0.05). The expression of BMP-2/SMAD1 signaling decreased in the shCCDC134 group, while it increased in the CCDC134 group (P < 0.05). Compared to the shCCDC134 group, osteogenic genes and proteins increased in the shCCDC134+BMP-2 group, and subcutaneous new bone formation increased in nude mice (P < 0.05). The indexes of the CCDC134+Dorsomorphin group decreased compared with the CCDC134 group (P < 0.05).Conclusion CCDC134 promotes the osteogenic differentiation of hDPSCs by regulating the BMP-2/SMAD1 signaling pathway.

Key words: dental pulp stem cells, CCDC134, osteogenic differentiation, tissue engineering, bone morphogenetic protein-2, recombinant human mothers against decapentaplegic homolog 1, Runt-related transcription factor 2, osteocalcin

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