Prrx1+牙周膜干细胞在牙移动过程中的谱系示踪

doi:10.3969/j.issn.1674-8115.2022.08.005

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (8): 1008-1015.doi: 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¹(), 金安婷¹(), 黄湘如¹, 徐弘远¹, 高昕¹, 杨屹羚¹, 代庆刚², 江凌勇¹()

^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

收稿日期:2022-04-14 接受日期:2022-08-13 出版日期:2022-08-28 发布日期:2022-10-08
通讯作者: 江凌勇 E-mail:838657340@qq.com;Katerinajin@foxmail.com;jianglingyong@sjtu.edu.cn
作者简介: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）
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)

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

WANG Xijun¹(), JIN Anting¹(), HUANG Xiangru¹, XU Hongyuan¹, GAO Xin¹, YANG Yiling¹, DAI Qinggang², JIANG Lingyong¹()

^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

Received:2022-04-14 Accepted:2022-08-13 Online:2022-08-28 Published:2022-10-08
Contact: JIANG Lingyong E-mail:838657340@qq.com;Katerinajin@foxmail.com;jianglingyong@sjtu.edu.cn
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)

摘要/Abstract

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-Cre^ERT2 小鼠与R26^tdTomato 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-Cre^ERT2；R26^tdTomato。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-Cre^ERT2；R26^tdTomato 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

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-Cre^ERT2^{mice were mated with R26^tdTomato 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-Cre^ERT2;R26^tdTomato . 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-Cre^ERT2;R26^tdTomato 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

中图分类号:

R783.5

引用本文

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.
WANG Xijun, JIN Anting, HUANG Xiangru, XU Hongyuan, GAO Xin, YANG Yiling, DAI Qinggang, JIANG Lingyong. Study on Prrx1⁺ periodontal ligament stem cells during orthodontic tooth movement by lineage tracing[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(8): 1008-1015.

使用本文

0

    /
/
推荐

导出引用管理器 EndNote|Ris|BibTeX

链接本文:
https://xuebao.shsmu.edu.cn/CN/10.3969/j.issn.1674-8115.2022.08.005



https://xuebao.shsmu.edu.cn/CN/Y2022/V42/I8/1008}}

图/表 5

表 1 PCR引物序列

Tab 1 Primer sequences for PCR

Gene	Forward sequence （5'→3'）	Reverse sequence （5'→3'）
Prrx1-Cre^ERT2	CGATGCAACGAGTGATGAGG	CGCATA ACCAGTGAAACAGC
tdTomato	TTCGGCTTCTGGCGTGTGAC	GAAGAGGAGTGCTCCGATCAGG
tdTomato-WT	AGCACTTGCTCTCCCAAAGTCGCTC	AAACTTCCCGACAAAACCGAA

图 1 PCR检测子代小鼠基因型Note：NC—negative control; "+"—positive control. TD means tdTomato strips; WT means wild-type strips.

Fig 1 Detection of genotype of offspring mice by PCR

图2 子代小鼠OTM模型的构建及验证Note：A. Construction of OTM model in offspring mice. The left side is the OTM side and the right side is the control side. B. Changes and statistics of tooth movement distance of offspring mice on OTM 3 d and OTM 7 d. The arrows indicate the direction of tooth movement.

Fig 2 Construction and validation of OTM model in offspring mice

图 3 压力区牙周膜的改变及 Prrx1+ 细胞谱系的表达Note：A. Observation of the compression area of the maxillary first molar in mice by low power microscope (×4). DB—distal buccal root; DP—distal palatine root; M—mesial root. The box shows the compression area. The arrow represents the direction of tooth movement. B. Observation of H-E staining of the periodontal ligament in the compression area of the Ctrl side and OTM side of mice by high power microscope (×40). AB—alveolar bone; D—dentin; PDL—periodontal ligament. C. Distribution of tdTomato + cells in the compression area of the Ctrl side and OTM side of mice by immunofluorescence staining (×40). D. Statistical analysis of the proportion of tdTomato + cells in the compression area of the Ctrl side and OTM side of mice. ns—no significance.

Fig 3 Changes of periodontal ligament and expression of Prrx1+ cell lineage in the compression area

图 4 张力区牙周膜的改变及 Prrx1+ 细胞谱系的表达Note：A. Observation of the tension area of the maxillary first molar in mice by low power microscope (×4). DB—distal buccal root; DP—distal palatine root; M—mesial root. The box shows the tension area. The arrow represents the direction of tooth movement. B. Observation of H-E staining of the periodontal ligament in the tension area of the Ctrl side and OTM side of mice by high power microscope (×40). AB—alveolar bone; D—dentin; PDL—periodontal ligament. C. Distribution of tdTomato + cells in the tension area of the Ctrl side and OTM side of mice by immunofluorescence staining (×40). D. Statistical analysis of the proportion of tdTomato + cells in the tension area of the Ctrl side and OTM side of mice. ns—no significance.

Fig 4 Changes of periodontal ligament and expression of Prrx1+ cell lineage in the tension area

参考文献 27

1	LI Y N, JACOX L A, LITTLE S H, et al. Orthodontic tooth movement: the biology and clinical implications[J]. Kaohsiung J Med Sci, 2018, 34(4): 207-214.
2	SEO B M, MIURA M, GRONTHOS S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament[J]. Lancet, 2004, 364(9429): 149-155.
3	WISE G E, KING G J. Mechanisms of tooth eruption and orthodontic tooth movement[J]. J Dent Res, 2008, 87(5): 414-434.
4	赵闫, 欧阳宁鹃, 代杰文, 等. 细胞谱系示踪技术及其在口腔医学研究中的应用进展[J].口腔生物医学, 2017, 8(4): 205-210.
	ZHAO Y, OUYANG N J, DAI J W, et al. Development of cell lineage tracing technology and its application in stomatology research[J]. Oral Biomed, 2017, 8(4): 205-210.
5	BULTE J W M, DOUGLAS T, WITWER B, et al. Monitoring stem cell therapy in vivo using magnetodendrimers as a new class of cellular MR contrast agents[J]. Acad Radiol, 2002, 9(Suppl 2): S332-S335.
6	NAGY A. Cre recombinase: the universal reagent for genome tailoring[J]. Genesis, 2000, 26(2): 99-109.
7	SHI Y, HE G X, LEE W C, et al. Gli1 identifies osteogenic progenitors for bone formation and fracture repair[J]. Nat Commun, 2017, 8(1): 2043.
8	YI Y, STENBERG W, LUO W, et al. Alveolar bone marrow Gli1⁺ stem cells support implant osseointegration[J]. J Dent Res, 2022, 101(1): 73-82.
9	MIZOGUCHI T, PINHO S, AHMED J, et al. Osterix marks distinct waves of primitive and definitive stromal progenitors during bone marrow development[J]. Dev Cell, 2014, 29(3): 340-349.
10	KAWANAMI A, MATSUSHITA T, CHAN Y Y, et al. Mice expressing GFP and CreER in osteochondro progenitor cells in the periosteum[J]. Biochem Biophys Res Commun, 2009, 386(3): 477-482.
11	BASSIR S H, GARAKANI S, WILK K, et al. Prx1 expressing cells are required for periodontal regeneration of the mouse incisor[J]. Front Physiol, 2019, 10: 591.
12	GONG X Y, ZHANG H, XU X Q, et al. Tracing PRX1⁺ cells during molar formation and periodontal ligament reconstruction[J]. Int J Oral Sci, 2022, 14(1): 5.
13	ZHONG Z A, SUN W H, CHEN H Y, et al. Optimizing tamoxifen-inducible Cre/Loxp system to reduce tamoxifen effect on bone turnover in long bones of young mice[J]. Bone, 2015, 81: 614-619.
14	DAI Q G, ZHOU S R, ZHANG P, et al. Force-induced increased osteogenesis enables accelerated orthodontic tooth movement in ovariectomized rats[J]. Sci Rep, 2017, 7(1): 3906.
15	KUSUMBE A P, RAMASAMY S K, STARSICHOVA A, et al. Sample preparation for high-resolution 3D confocal imaging of mouse skeletal tissue[J]. Nat Protoc, 2015, 10(12): 1904-1914.
16	孙聪, 刘文佳, 金岩, 等. 小鼠正畸牙齿移动模型的制作[J]. 牙体牙髓牙周病学杂志, 2017, 27(5): 280-283.
	SUN C, LIU W J, JIN Y, et al. Establishment of mouse model of orthodontic tooth movement[J]. Chin J Conserv Dent, 2017, 27(5): 280-283.
17	ABDALLAH B M, KASSEM M. Human mesenchymal stem cells: from basic biology to clinical applications[J]. Gene Ther, 2008, 15(2): 109-116.
18	WANG K, XU C, XIE X, et al. Axin2⁺ PDL cells directly contribute to new alveolar bone formation in response to orthodontic tension force[J]. J Dent Res, 2022, 101(6): 695-703.
19	KIM H, KIM M, IM S K, et al. Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes[J]. Lab Anim Res, 2018, 34(4): 147-159.
20	FEIL R, BROCARD J, MASCREZ B, et al. Ligand-activated site-specific recombination in mice[J]. Proc Natl Acad Sci U S A, 1996, 93(20): 10887-10890.
21	XIE X, WANG J, WANG K, et al. Axin2⁺-mesenchymal PDL cells, instead of K14⁺ epithelial cells, play a key role in rapid cementum growth[J]. J Dent Res, 2019, 98(11): 1262-1270.
22	LI B, WANG Y G, FAN Y, et al. Cranial suture mesenchymal stem cells: insights and advances[J]. Biomolecules, 2021, 11(8): 1129.
23	MARTIN J F, BRADLEY A, OLSON E N. The paired-like homeo box gene MHox is required for early events of skeletogenesis in multiple lineages[J]. Genes Dev, 1995, 9(10): 1237-1249.
24	LU M F, CHENG H T, KERN M J, et al. Prx-1 functions cooperatively with another paired-related homeobox gene, prx-2, to maintain cell fates within the craniofacial mesenchyme[J]. Development, 1999, 126(3): 495-504.
25	MITCHELL J M, HICKLIN D M, DOUGHTY P M, et al. The Prx1 homeobox gene is critical for molar tooth morphogenesis[J]. J Dent Res, 2006, 85(10): 888-893.
26	BALIC A, ADAMS D, MINA M N. Prx1 and Prx2 cooperatively regulate the morphogenesis of the medial region of the mandibular process[J]. Dev Dyn, 2009, 238(10): 2599-2613.
27	WANG J, SARASWAT D, SINHA A K, et al. Paired related homeobox protein 1 regulates quiescence in human oligodendrocyte progenitors[J]. Cell Rep, 2018, 25(12): 3435-3450.e6.

Prrx1⁺牙周膜干细胞在牙移动过程中的谱系示踪

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

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 27

相关文章 1

编辑推荐

Metrics

Prrx1+牙周膜干细胞在牙移动过程中的谱系示踪

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

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 27

相关文章 1

编辑推荐

Metrics

Prrx1⁺牙周膜干细胞在牙移动过程中的谱系示踪

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