中国实用口腔科杂志

中国实用口腔科杂志 ›› 2022, Vol. 15 ›› Issue (1): 76-82.DOI: 10.19538/j.kq.2022.01.014

• 论著 • 上一篇    下一篇

外泌体诱导3D纳米纤维小管中牙髓干细胞成骨/成牙本质向分化的作用研究

梅笑寒1，仇    珺1，张贝蒂2，王珏钰1，余　擎1，张亚庆1，肖    敏1   

  1. 1. 军事口腔医学国家重点实验室，口腔疾病国家临床医学研究中心，陕西省口腔医学重点实验室，空军军医大学第三附属医院牙体牙髓病科，陕西  西安  710032；2. 苏州大学附属第一医院口腔科，江苏  苏州  215000
  • 出版日期:2022-01-30 发布日期:2022-03-03
  • 基金资助:
    国家自然科学基金（81870751）；陕西省自然科学基础研究计划（2021JM-235；2019JQ-282）

  • Online:2022-01-30 Published:2022-03-03

摘要： 目的    探究基质细胞衍生因子-1α（stromal cell-derived factor-1α，SDF-1α）诱导的根尖牙乳头干细胞来源外泌体（exosomes derived from stem cells from apical papilla，SCAP-Exo）对3D纳米纤维小管中牙髓干细胞（dental pulp stem cells，DPSCs）增殖和成骨/成牙本质向分化的影响。方法    提取SDF-1α诱导的SCAP-Exo，采用透射电镜、纳米粒子跟踪技术及Western Blot进行鉴定。将DPSCs培养于3D纳米纤维小管中，扫描电镜观察DPSCs的形态与黏附状态。使用不同质量浓度（0、50、100 μg/mL）的SCAP-Exo刺激3D纳米纤维小管中的DPSCs，分别记为对照组、50 μg/mL SCAP-Exo组和100 μg/mL SCAP-Exo组，并对细胞增殖活力和碱性磷酸酶（ALP）活性进行检测；实时RT-PCR和Western Blot分别检测成骨/成牙本质向分化相关基因的mRNA和蛋白表达水平。结果    SCAP-Exo形态呈茶托状，具有双层膜结构，平均粒径为126.4 nm，能够表达外泌体标志蛋白CD9和CD81。扫描电镜观察显示DPSCs在3D纳米纤维小管中的黏附状态良好。50 μg/mL SCAP-Exo组和100 μg/mL SCAP-Exo组DSPCs的增殖活力和ALP活性均高于对照组，且100 μg/mL SCAP-Exo组较50 μg/mL SCAP-Exo组的ALP活性升高更显著（均P < 0.05）。100 μg/mL SCAP-Exo组成骨/成牙本质向分化基因（ALP、DMP-1、BSP和OCN）的mRNA和蛋白表达水平均高于对照组（均P < 0.05）。结论    DPSCs可在3D纳米纤维小管中维持良好的增殖活力。SDF-1α诱导的SCAP-Exo可增强3D纳米纤维小管中DPSCs的增殖活力和ALP活性，以及促进其向成骨/成牙本质向分化。

关键词: 牙髓干细胞, 外泌体, 成骨/成牙本质向分化, 管状支架

Abstract: Objective    To explore the effect of stromal cell-derived factor-1α（SDF-1α）-induced exosomes derived from stem cells from apical papilla（SCAP-Exo）on the proliferation and osteo/odontogenic differentiation of dental pulp stem cells（DPSCs）cultured in 3D nanofiber tubules scaffold. Methods    SCAP-derived exosomes induced by SDF-1α were isolated and were identified by transmission electron microscopy，nanoparticle tracking analysis and Western Blot. DPSCs were cultured in 3D nanofiber tubules scaffold，and scanning electron microscopy was used to observe the morphology and adhesion of DPSCs. DPSCs cultured in 3D nanofiber tubules scaffold were stimulated with different mass concentrations of SCAP-Exo（0，50，100 μg/mL），which were respectively recorded as control group，50 μg/mL SCAP-Exo group，and 100 μg/mL SCAP-Exo group，and the cell proliferation and alkaline phosphatase（ALP）activity of DPSCs were detected. Real-time RT-PCR and Western Blot assay were used to evaluate the gene mRNA and protein level related to osteo/odontogenic differentiation of DPSCs cultured in 3D nanofiber tubules scaffold. Results    The bilayer membrane and cup-shaped appearance of representative exosomes were observed. The average size of SCAP-Exo was at 126.4 nm. The exosomes expressed exosomal marker proteins CD9 and CD81. The SEM observed that DPSCs grew and adhered well in the 3D nanofiber tubules scaffold. The proliferation activity and ALP activity of DSPCs in the 50 μg/mL SCAP-Exo group and 100 μg/mL SCAP-Exo group were increased compared with the control group，and the ALP activity of the 100 μg/mL SCAP-Exo group increased more significantly than that of the 50 μg/mL SCAP-Exo group（P < 0.05）. The expression levels of mRNA and proteins of the osteo/odontogenic differentiation-related genes （ALP，DMP-1，BSP and OCN） in 100 μg/mL SCAP-Exo group were higher than the control group（P < 0.05）. Conclusion    DPSCs can maintain good proliferation viability in 3D nanofibrous tubules scaffold，and SDF-1α-induced SCAP-Exo can increase the proliferation and ALP activity of DPSCs cultured in 3D nanofibrous tubules scaffold，and promote the osteo/odontogenic differentiation. 

Key words: dental pulp stem cells, exosome, osteo/odontogenic differentiation, tubular scaffold