上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2022 , Vol. 42 >Issue 8: 1008 - 1015

DOI: https://doi.org/10.3969/j.issn.1674-8115.2022.08.005

论著 · 基础研究

Prrx1+牙周膜干细胞在牙移动过程中的谱系示踪

  • 汪席均 ,
  • 金安婷 ,
  • 黄湘如 ,
  • 徐弘远 ,
  • 高昕 ,
  • 杨屹羚 ,
  • 代庆刚 ,
  • 江凌勇
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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外科，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 200011
    2.上海交通大学医学院附属第九人民医院口腔第二门诊部，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 200011
汪席均（1996—），女，硕士生；电子信箱：838657340@qq.com
金安婷（1995—），女，硕士；电子信箱：Katerinajin@foxmail.com第一联系人：（汪席均、金安婷并列第一作者）
江凌勇，电子信箱：jianglingyong@sjtu.edu.cn。

收稿日期: 2022-04-14

  录用日期: 2022-08-13

  网络出版日期: 2022-10-08

基金资助

国家自然科学基金(82071083);上海市自然科学基金(22ZR1436700);上海市优秀学术/技术带头人计划(20XD1422300);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附属第九人民医院生物样本库项目(YBKB201919);上海申康医院发展中心临床创新三年行动计划(SHDC2020CR4084);上海交通大学医学院大学生创新训练计划项目(S202110248114)

收起

Study on Prrx1+ periodontal ligament stem cells during orthodontic tooth movement by lineage tracing

  • Xijun WANG ,
  • Anting JIN ,
  • Xiangru HUANG ,
  • Hongyuan XU ,
  • Xin GAO ,
  • Yiling YANG ,
  • Qinggang DAI ,
  • Lingyong JIANG
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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 200011, China
    2.The 2nd Dental Centre, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology, Shanghai 200011, China
JIANG Lingyong, E-mail: jianglingyong@sjtu.edu.cn.

Received date: 2022-04-14

  Accepted date: 2022-08-13

  Online published: 2022-10-08

Supported by

National Natural Science Foundation of China(82071083);Natural Science Foundation of Shanghai(22ZR1436700);Program of Shanghai Academic/Technology Research Leader(20XD1422300);Cross-disciplinary Research Fund of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine(JYJC202116);Project of Biobank of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine(YBKB201919);Clinical Research Plan of Shanghai Hospital Development Center(SHDC2020CR4084);Students Innovation Training Program of Shanghai Jiao Tong University School of Medicine(S202110248114)

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摘要

目的·通过Cre/loxP重组酶系统研究牙周膜干细胞（periodontal ligament stem cells，PDLSCs）中配对相关同源基因1（paired related homeobox 1，Prrx1）阳性细胞谱系（Prrx1+细胞谱系）在小鼠牙移动过程中的动态分布。方法·利用Cre/loxP重组酶系统，将诱导型Prrx1-CreERT2 小鼠与R26tdTomato 荧光标记小鼠交配繁殖，并通过PCR对其子代小鼠进行基因型鉴定。取8只子代小鼠，向其腹腔注射他莫昔芬（tamoxifen，TA），以标记PDLSCs中的Prrx1+细胞谱系（即tdTomato+细胞）。于小鼠上颌左侧安置加力装置［即正畸牙移动（orthodontics tooth movement，OTM）侧］，右侧未加力即为对照侧，以构建小鼠OTM模型。分别于牙移动第3日（OTM 3 d）、第7日（OTM 7 d）时处死小鼠，收集其双侧上颌磨牙及周围牙周组织，经脱钙后行包埋及冰冻切片处理。采用苏木精-伊红染色（hematoxylin-eosin staining，H-E染色）观察张力区和压力区牙周膜的改变，并利用免疫荧光染色观察Prrx1+细胞谱系在张力区及压力区的动态分布。结果·经PCR鉴定，子代小鼠基因型为Prrx1-CreERT2；R26tdTomato。随着加力装置作用的增加，OTM 7 d小鼠的牙移动距离［（87.44±4.02）μm］较OTM 3 d小鼠［（42.81±5.04）μm］明显增加，提示小鼠OTM模型构建成功。H-E染色结果显示：OTM 3 d时小鼠OTM侧压力区的牙周膜被压缩、间隙变窄，而OTM 7 d时OTM侧牙周膜宽度逐渐恢复；OTM 3 d时OTM侧张力区的牙周膜被拉伸，而OTM 7 d较OTM 3 d时牙周膜排列更规则。免疫荧光染色结果显示：OTM 3 d时OTM侧压力区牙周膜中tdTomato+细胞数量较对照侧减少，OTM 7 d时OTM侧压力区tdTomato+细胞数量较对照侧增加（均P<0.05）；OTM 3 d及OTM 7 d时，OTM侧张力区tdTomato+细胞数量较对照侧均有增加（均P<0.05）。结论·成功构建了Prrx1-CreERT2；R26tdTomato 小鼠的OTM模型，并初步证实了Prrx1+细胞谱系参与OTM过程中的牙周改建。

关键词： 细胞谱系示踪; 正畸牙移动; 配对相关同源基因1; Cre/loxP重组酶系统

本文引用格式

汪席均 , 金安婷 , 黄湘如 , 徐弘远 , 高昕 , 杨屹羚 , 代庆刚 , 江凌勇 . Prrx1+牙周膜干细胞在牙移动过程中的谱系示踪[J]. 上海交通大学学报（医学版）, 2022 , 42(8) : 1008 -1015 . DOI: 10.3969/j.issn.1674-8115.2022.08.005

Abstract

Objective ·To investigate the dynamic distribution of paired related homebox 1 (Prrx1) positive cell lineage (Prrx1+ cell lineage) in periodontal ligament stem cells (PDLSCs) during tooth movement in mice by Cre/loxP recombination system. Methods ·By using Cre/loxP recombination system, inducible Prrx1-CreERT2mice were mated with R26tdTomato fluorescently labeledmice, and their offspring mice were genotyped by PCR. Eight offspring mice were injected with tamoxifen (TA) intraperitoneally to mark the Prrx1+cell lineage (tdTomato+ cells) in PDLSCs. The orthodontics tooth movement (OTM) model was constructed by placing a force-loading device on the left side of maxilla (i.e., OTM side), and the right side without force was control side (i.e., Ctrl side). The mice were sacrificed on the third day (OTM 3 d) and the seventh day (OTM 7 d) of tooth movement, and their bilateral maxillary molars and surrounding periodontium were collected, decalcified, embedded and frozen sectioned. The changes of periodontal ligament in the tension area and compression area were observed by hematoxylin-eosin staining (H-E staining), and the dynamic distribution of Prrx1+ celllineage was observed by immunofluorescence staining. Results ·The genotype of the offspring mice was identified by PCR as Prrx1-CreERT2;R26tdTomato . With the increased action of the stressing device, the tooth movement distance of OTM 7 d mice [(87.44±4.02) μm] increased significantly compared with that of the OTM 3 d mice [(42.81±5.04) μm], suggesting OTM model was successfully constructed. H-E staining showed that the periodontal ligament and its gap in the compression area of the OTM side was compressed and narrowed on OTM 3 d, and its width was gradually restored on OTM 7 d; the periodontal ligament in the tension area of the OTM side was stretched on OTM 3 d, and the periodontal ligament was more regularly arranged on OTM 7 d than that on OTM 3 d. Immunofluorescence staining showed that the number of tdTomato+ cells in the periodontal ligament of the compression area on the OTM side was lower than that of the Ctrl side on OTM 3 d, and the number of tdTomato+ cells in compression area on the OTM side was higher than that of the Ctrl side on OTM 7 d (both P<0.05); on both OTM 3 d and OTM 7 d, the number of tdTomato+ cells in tension area on the OTM side increased compared with that on the Ctrl side (both P<0.05). Conclusion ·The OTM model of Prrx1-CreERT2;R26tdTomato mice is successfully constructed, and the involvement of Prrx1+ cell lineage in periodontal remodeling during OTM is tentatively confirmed.

Key words： cell lineage tracing; orthodontic tooth movement (OTM); paired related homeobox1 (Prrx1); Cre/loxP recombinase system

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