Effects of Necdin on odontogenic differentiation and mineralization of rat dental germ ectodermal mesenchymal stem cells
- Journal of Army Medical University Vol. 45, Issue 13, Pages: 1413-1421(2023)
- 作者机构：
646000 四川 泸州，西南医科大学附属口腔医院正畸科，口颌面修复重建和再生泸州市重点实验室，西南医科大学口腔医学研究所
- 作者简介：
- 基金信息：the General Program of National Natural Science Foundation of China(81970906);the Project of Science and Technology Plan of Sichuan Province(2019YJ0689)
DOI：10.16016/j.2097-0927.202303095
CLC： R322.41;R329.21;R341Received：15 March 2023，
Revised：2023-04-10，
Published：15 July 2023
- 稿件说明：
移动端阅览
Yixin ZHANG, Hongyan YUAN, Mingjie LU, et al. Effects of Necdin on odontogenic differentiation and mineralization of rat dental germ ectodermal mesenchymal stem cells[J]. Journal of Army Medical University, 2023, 45(13): 1413-1421.
Yixin ZHANG, Hongyan YUAN, Mingjie LU, et al. Effects of Necdin on odontogenic differentiation and mineralization of rat dental germ ectodermal mesenchymal stem cells[J]. Journal of Army Medical University, 2023, 45(13): 1413-1421. DOI： 10.16016/j.2097-0927.202303095.
摘要
目的
2
探讨胚胎癌细胞来源分化神经元特异蛋白(neurally differentiated EC-cell-derived protein，Necdin)对大鼠牙胚外胚间充质干细胞(ectomesenchymal stem cells，EMSCs)成牙分化和矿化的调控作用。
方法
2
利用免疫荧光染色检测Necdin在E13.5d、E19.5d大鼠牙胚中的表达分布情况。分离培养E19.5d大鼠牙胚EMSCs，流式细胞术鉴定细胞表型，构建Necdin沉默慢病毒感染EMSCs，分为阴性对照组(sh-NC)和沉默组(sh-Necdin)。流式细胞术检测EMSCs凋亡情况，CCK-8法检测EMSCs增殖能力，划痕实验检测EMSCs迁移能力，矿化诱导后通过碱性磷酸酶染色、茜素红染色、RT-PCR和Western blot观察Necdin对EMSCs成牙分化和矿化的调控作用。
结果
2
免疫荧光显示Necdin在内、外釉上皮和上皮与间充质接触区域呈强阳性表达；牙胚组织来源的EMSCs沉默Necdin后，sh-Necdin组EMSCs凋亡率较sh-NC组增多(
P
＜0.01)，与sh
-NC组相比，sh-Necdin组EMSCs的增殖能力更强(
P
＜0.01)，但迁移能力变弱(
P
＜0.01)；与sh-NC组相比，sh-Necdin组碱性磷酸酶染色更浅且矿化结节形成更少；sh-Necdin组EMSCs成牙分化和矿化相关的基因与蛋白表达量明显低于sh-NC组(
P
＜0.05)。
结论
2
Necdin在大鼠牙胚未来成牙分化和矿化区域呈特异性表达，沉默Necdin促进EMSCs凋亡和增殖，但抑制EMSCs迁移以及成牙分化和矿化，Necdin可能在牙发育矿化过程中发挥重要作用。
Abstract
Objective
2
To investigate the regulative effect of neurally differentiated EC-cell-derived protein
Necdin
on the odontogenic differentiation and mineralization of rat dental germ ectomesenchymal stem cells (EMSCs).
Methods
2
Immunofluorescence staining was used to detect the expression and distribution of Necdin in E13.5d and E19.5d rat dental germ. EMSCs derived from E19.5d rat dental germ were isolated and cultured
and identified with flow cytometry for cell surface antigens. The EMSCs were stably transfected with lentiviral vector of Necdin silencing. Then
the apoptosis of EMSCs was detected by flow cytometry in the negative control group (sh-NC) and silenced group (sh-Necdin). The proliferation capacity of EMSCs was detected by CCK-8 assay
and the migration ability of EMSCs was detected by cell scratching assay. After mineralization induction
the regulative effect of Necdin on the odontogenic differentiation and mineralization of EMSCs was observed by alkaline phosphatase staining
alizarin red staining
RT-PCR and Western blotting.
Results
2
The immunofluorescence results showed that Necdin was strongly expressed and concentrated in the inner enamel epithelium
outer enamel epithelium and in the contact area between epithelium and mesenchyme. After Necdin silencing
the EMSCs showed higher apoptotic rate
enhanced proliferation ability but weaker migration ability when compared with the cells from the sh-NC group (all
P
<
0.01). What's more
Necdin silencing also resulted in lighter alkaline phosph
atase stain and less mineralized nodule formation
and up-regulations of the molecules related to tooth differentiation and mineralization at mRNA and protein levels (
P
<
0.05) in the cells from the sh-Necdin group than those from the sh-NC group.
Conclusion
2
Necdin is specifically expressed in the future odontogenic differentiation and mineralization regions in rat dental germ. Its silencing promotes the apoptosis and proliferation
but inhibits the migration and the abilities of odontogenesis differentiation and mineralization in EMSCs. Necdin might play an important role in tooth development and mineralization.
关键词
Keywords
references
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