上海交通大学学报（医学版）

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2024 , Vol. 44 >Issue 9: 1083 - 1093

DOI: https://doi.org/10.3969/j.issn.1674-8115.2024.09.003

论著 · 基础研究

全反式视黄酸调控颌骨骨髓间充质干细胞成骨分化双向效应的体外研究

  • 刘媛琪 ,
  • 孙思远 ,
  • 代庆刚 ,
  • 江凌勇 ,
  • 沈国芳
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  • 1.上海交通大学医学院附属第九人民医院口腔颅颌面科，上海交通大学口腔医学院，国家口腔医学中心，国家口腔疾病临床医学研究中心，上海市口腔医学重点实验室，上海市口腔医学研究所，上海 200011
    2.上海 -新西兰口腔医学联合实验室-口腔颅颌面基础与临床研究中心，上海交通大学医学院附属第九人民医院，上海 200011
    3.上海交通大学医学院附属第九人民医院口腔第二门诊部，上海交通大学口腔医学院，国家口腔医学中心，国家口腔疾病临床医学研究中心，上海市口腔医学重点实验室，上海市口腔医学研究所，上海 200011
刘媛琪（1996—），女，回族，硕士生；电子信箱：liuyuanqi@sjtu.edu.cn
孙思远（1998—），男，硕士生；电子信箱：ssy1412832053@163.com。第一联系人：（刘媛琪、孙思远并列第一作者）
沈国芳，电子信箱：shengf1428@sjtu.edu.cn。

收稿日期: 2024-04-03

  录用日期: 2024-04-25

  网络出版日期: 2024-09-28

基金资助

国家自然科学基金(82071083);中央高校基本科研业务费专项资金(YG2023ZD14);上海市自然科学基金(21ZR1436900);上海市科技创新行动计划国际科技合作项目/政府间国际科技合作项目(23410713600);上海交通大学医学院附属第九人民医院交叉研究基金(JYJC202116);上海交通大学医学院生物材料与再生医学交叉研究项目(2022LHB02);上海交通大学医学院“双百人”项目(20221809);上海交通大学医学院附属第九人民医院原创项目(JYYC003)

收起

Dual-directional effect of all-trans retinoic acid on osteogenic differentiation of jaw bone marrow mesenchymal stem cells in vitro

  • Yuanqi LIU ,
  • Siyuan SUN ,
  • Qinggang DAI ,
  • Lingyong JIANG ,
  • Guofang SHEN
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  • 1.Department of Oral and Maxillofacial Surgery, Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology; Shanghai Research Institute of Stomatology, Shanghai 200011, China
    2.Shanghai -New Zealand Joint Laboratory of Dentistry, Centre for Clinical and Fundamental Craniofacial Research, Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200011, China
    3.Department of 2nd Dental Centre, Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology; Shanghai Research Institute of Stomatology, Shanghai 200011, China
SHEN Guofang, E-mail: shengf1428@sjtu.edu.cn.

Received date: 2024-04-03

  Accepted date: 2024-04-25

  Online published: 2024-09-28

Supported by

National Natural Science Foundation of China(82071083);The Fundamental Research Funds for the Central Universities(YG2023ZD14);Natural Science Foundation of Shanghai(21ZR1436900);Shanghai Science and Technology Innovation Action Plan-International Science and Technology Cooperation Program(23410713600);Cross-Disciplinary Research Fund of Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine(JYJC202116);Biomaterials and Regenerative Medicine Institute Cooperative Research Project of Shanghai Jiao Tong University School of Medicine(2022LHB02);“Two-Hundred Talents” Program of Shanghai Jiao Tong University School of Medicine(20221809);Original Exploration Project of Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine(JYYC003)

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摘要

目的·探究不同浓度全反式视黄酸（all-trans retinoic acid，ATRA）对大鼠颌骨骨髓间充质干细胞（jaw bone marrow mesenchymal stem cells，jBMSCs）成骨分化的影响。方法·通过全骨髓贴壁法分离培养4周龄Sprague-Dawley（SD）大鼠jBMSCs。运用流式细胞术鉴定jBMSCs表面抗原。利用碱性磷酸酶（alkaline phosphatase，ALP）染色/茜素红染色、油红O染色及阿尔辛蓝染色分别对成骨诱导、成脂诱导及成软骨诱导后的jBMSCs进行多向分化潜能检测。分别使用ATRA浓度为0.01、0.1、1、5、10、20 μmol/L的成骨诱导液对jBMSCs进行体外成骨诱导，并使用二甲基亚砜（dimethyl sulfoxide，DMSO）作为对照组，利用CCK8进行细胞活力检测。采用ALP染色和茜素红染色对各浓度组jBMSCs的成骨分化能力进行检测，并筛选后续实验浓度。利用实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction，qPCR）、免疫荧光染色分析不同浓度ATRA下jBMSCs成骨相关基因mRNA与蛋白表达水平。结果·流式细胞术分析显示，98%以上的P1代jBMSCs表现为CD29+CD90+CD31-CD45-，与骨髓间充质干细胞表面抗原特点相符。ALP染色/茜素红染色、油红O染色及阿尔辛蓝染色结果证明P1代jBMSCs具有成骨、成脂、成软骨多向分化能力。ALP染色/茜素红染色结果显示，0.01、0.1和1 μmol/L ATRA组jBMSCs成骨活性和矿化能力较对照组增强，而继续提升ATRA浓度则使成骨活性与矿化能力减弱，浓度高于5 μmol/L时开始低于对照组水平（均P<0.05）。qPCR分析发现，0.1、1 μmol/L ATRA组成骨相关基因如Alp、骨唾液酸蛋白（bone sialoprotein，Bsp）、Ⅰ型胶原蛋白α1（collagen type Ⅰ α1，Col1a1）、骨钙素（osteocalcin，Ocn）表达水平较对照组上升，而继续提升ATRA浓度则使基因表达水平下降，ATRA浓度高于5 μmol/L时开始低于对照组水平（均P<0.05）。免疫荧光染色结果显示，0.1、1 μmol/L ATRA组较对照组成骨相关蛋白成骨细胞特异性转录因子SP7、ALP及OCN表达增强，而继续提升ATRA浓度则使蛋白表达下降，浓度高于5 μmol/L时开始低于对照组水平（均P<0.05）。结论·较低浓度（0.1、1 μmol/L）ATRA可促进大鼠jBMSCs成骨分化能力，且该促进效应在0.1 μmol/L浓度时达到峰值，进一步提升浓度可使该促进效应减弱。较高浓度（5、10、20 μmol/L）ATRA对大鼠jBMSCs成骨分化呈现抑制效应。研究在体外证明ATRA对大鼠jBMSCs成骨分化具双向效应，并鉴定了0.1 μmol/L ATRA为大鼠jBMSCs成骨分化的最适浓度，为体内研究的开展与全反式视黄酸的临床应用提供了参考依据。

关键词： 全反式视黄酸; 颌骨; 骨髓间充质干细胞; 成骨分化; 双向效应

本文引用格式

刘媛琪 , 孙思远 , 代庆刚 , 江凌勇 , 沈国芳 . 全反式视黄酸调控颌骨骨髓间充质干细胞成骨分化双向效应的体外研究[J]. 上海交通大学学报（医学版）, 2024 , 44(9) : 1083 -1093 . DOI: 10.3969/j.issn.1674-8115.2024.09.003

Abstract

Objective ·To explore the effect of all-trans retinoic acid (ATRA) of different concentrations on osteogenic differentiation of jaw bone mesenchymal stem cells (jBMSCs) in rats. Methods ·jBMSCs from 4-week-old Sprague-Dawley (SD) rats were isolated and cultured with whole bone marrow adherence method. The surface antigens were identified by using flow cytometry. Alkaline phosphatase (ALP) staining/alizarin red staining, oil red O staining and alcian blue staining were used to prove the multilineage differentiation potential of jBMSCs after osteogenic, adipogenic and chondrogenic induction respectively. jBMSCs were induced in osteogenic medium with ATRA of concentration of 0.01, 0.1, 1, 5, 10, 20 μmol/L in vitro, and dimethyl sulfoxide (DMSO) was used as control group. Cell viability of jBMSCs in different groups were determined by CCK8. ALP staining and alizarin red staining were used to investigate the osteogenic ability of jBMSCs in each group and screened the concentrations for subsequent experiments. Quantitative real-time polymerase chain reaction (qPCR) and immunofluorescence staining were used to analyze the expressions of osteogenesis-related genes and proteins in jBMSCs of different concentrations. Results ·The flow cytometry analysis showed that more than 98% of P1 jBMSCs were positive for CD29+CD90+CD31-CD45-, which was congruent with the characteristics of bone mesenchymal stem cells. The results of ALP staining/alizarin red staining, oil red O staining and alcian blue staining indicated that the P1 jBMSCs had the multilineage differentiation potential of osteogenesis, adipogenesis and chondrogenesis. The results of ALP staining/alizarin red staining showed that the osteogenic activity and mineralization ability of jBMSCs in 0.01, 0.1 and 1 μmol/L ATRA groups were increased compared with those in the control group, while the osteogenic activity and mineralization ability were decreased when the concentration of ATRA increased, especially higher than 5 μmol/L (all P<0.05). qPCR analysis showed that the mRNA expression levels of osteogenesis-related genes such as Alp, bone sialoprotein (Bsp), collagen type Ⅰ α1 (Col1a1) and osteocalcin (Ocn) were higher in the 0.1 and 1 μmol/L ATRA groups compared to the control group. However, further increasing the concentration of ATRA led to a decrease in gene expression levels, and when the concentration exceeded 5 μmol/L, it began to be lower than the control group level (all P<0.05). The immunofluorescence staining showed that the expression of osteogenic related proteins SP7, ALP and OCN in the 0.1 and 1 μmol/L ATRA groups were increased compared to the control group, while further increasing the concentration of ATRA led to a decrease in protein expression. When the concentration was higher than 5 μmol/L, it began to be lower than the control group level (all P<0.05). Conclusion ·Lower concentrations (0.1, 1 μmol/L) of ATRA can promote the osteogenic differentiation of rat jBMSCs, and the promoting effect reaches its peak at 0.1 μmol/L, while the effect can be weakened by further increasing the concentration. Higher concentrations (5, 10, 20 μmol/L) of ATRA could inhibit the osteogenic differentiation of rat jBMSCs, showing an inhibitory effect. In this study, the dual-directional effect of retinoic acid on osteogenic differentiation of jBMSCs was demonstrated in vitro, and 0.1 μmol/L ATRA was identified as the optimal concentration for osteogenic differentiation of jBMSCs in rats, which provided a reference basis for the development of in vivo studies and clinical application of ATRA.

Key words： all-trans retinoic acid (ATRA); jaw; bone marrow mesenchymal stem cell; osteogenic differentiation; dual-directional effect

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