中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (25): 4004-4010.doi: 10.3969/j.issn.2095-4344.1787

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

miR-210促进大鼠牙髓干细胞的增殖和牙源性分化

王艳玲，左春然，王  静，杨  琨，王守儒，张小平
  

  1. 河南省中医院(河南中医药大学第二附属医院)口腔科，河南省郑州市  450002
  • 修回日期:2019-04-10 出版日期:2019-09-08 发布日期:2019-09-08
  • 通讯作者: 王艳玲，硕士，主治医师，河南省中医院(河南中医药大学第二附属医院)口腔科，河南省郑州市 450002
  • 作者简介:王艳玲，女，1979年生，河南省虞城县人，汉族，2014年河北联合大学毕业，硕士，主治医师，主要从事口腔颌面外科及种植研究。
  • 基金资助:

    河南省科技厅省部级课题(172102310613)，项目负责人：王艳玲

MicroRNA-210 promotes the proliferation and odontogenic differentiation of rat dental pulp stem cells

Wang Yanling, Zuo Chunran, Wang Jing, Yang Kun, Wang Shouru, Zhang Xiaoping
  

  1. Department of Stomatology, Henan Traditional Chinese Medicine Hospital (Second Affiliated Hospital of Henan University of Chinese Medicine), Zhengzhou 450002, Henan Province, China
  • Revised:2019-04-10 Online:2019-09-08 Published:2019-09-08
  • Contact: Wang Yanling, Department of Stomatology, Henan Traditional Chinese Medicine Hospital (Second Affiliated Hospital of Henan University of Chinese Medicine), Zhengzhou 450002, Henan Province, China
  • About author:Wang Yanling, Master, Attending physician, Department of Stomatology, Henan Traditional Chinese Medicine Hospital (Second Affiliated Hospital of Henan University of Chinese Medicine), Zhengzhou 450002, Henan Province, China
  • Supported by:

    the Project of Henan Provincial Science and Technology Department, No. 172102310613 (to WYL)

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摘要：

文章快速阅读：

文题释义：
牙髓干细胞的分化：牙髓干细胞是存在于牙髓中的具有间充质干细胞特性的成体干细胞，衍生于胚胎时期的外胚层细胞，具有增殖活性高、免疫原性低、可塑性强等优点。研究已证实，牙髓干细胞经诱导可定向分化为脂肪细胞、神经细胞、软骨细胞、心肌细胞、成骨细胞和成牙本质细胞等，在牙周组织工程方面有巨大潜力。
MiR-210：miRNA是广泛存在于真核生物细胞内的由多个核苷酸组成的单链小RNA，参与和调控生物体的生长发育及组织分化，在信号转导和疾病发生中发挥决定性作用。miRNA参与调节干细胞关键基因的表达，维持干细胞的自我更新和多向分化潜能。以往的研究证实，miR-210可能调控牙髓干细胞的成牙本质分化和成骨分化，但只停留在miRNA基因芯片技术检测miRNA在牙髓干细胞中的表达，尚未有miR-210在牙髓干细胞分化中作用的实验研究报道。

 

摘要
背景：miRNA表达谱预测miR-210可能在牙髓干细胞向牙本质和成骨方向分化中发挥作用，但具体作用尚不明确。
目的：探讨miR-210在大鼠牙髓干细胞增殖和牙源性分化中的作用。
方法：经河南中医药大学第二附属医院伦理委员会批准，收集5只SD大鼠牙髓组织，分离获得牙髓干细胞并分为5组：正常对照组、miR-210模拟物阴性对照组、miR-210模拟物组、miR-210抑制物阴性对照组和miR-210抑制物组。实时荧光定量PCR和Western blot检测细胞中miR-210、ALP、DSPP、DMP-1、GIT2、OPN mRNA的表达和DSPP、DMP-1、OPN和OCN蛋白的表达，CCK-8细胞增殖检测试剂盒检测细胞的增殖能力，比色法检测细胞碱性磷酸酶活性，茜素红染色检测矿物质合成能力。
结果与结论：①miR-210模拟物组细胞的增殖活力高于其他4组，差异有显著性意义(P < 0.01)；miR-210抑制物组细胞的增殖活力低于其他4组，差异有显著性意义(P < 0.01)；②miR-210模拟物组细胞的碱性磷酸酶活性高于其他4组，差异有显著性意义(P < 0.01)；miR-210抑制物组细胞的碱性磷酸酶活性低于其他4组，差异有显著性意义(P < 0.01)；③茜素红染色结果显示，miR-210模拟物组细胞表面布满钙盐沉积形成的大小不等的暗红色结节，数量多于其他4组，miR-210抑制物组细胞表面的暗红色钙结节散在分布，数量少于其他4组；④miR-210模拟物组细胞中miR-210、ALP、DSPP、DMP1、GIT2、OPN mRNA和DSPP、DMP1、OPN、OCN蛋白的表达高于其他4组，差异有显著性意义(P < 0.01)；miR-210抑制物组细胞中miR-210、ALP、DSPP、DMP1、GIT2、OPN mRNA和DSPP、DMP1、OPN、OCN蛋白的表达低于其他4组，差异有显著性意义(P < 0.01)；⑤结果提示，miR-210能够促进大鼠牙髓干细胞增殖与牙源性分化。


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID: 0000-0003-1394-4695(王艳玲)

关键词: 牙髓干细胞, miRNA, miR-210, 牙源性分化, 细胞增殖, 碱性磷酸酶活性, 矿物质合成能力

Abstract:

BACKGROUND: MicroRNAs profile predicts that microRNAs-210 may play a role in the differentiation of dental pulp stem cells into dentin and osteogenesis, but the specific role remains unclear.
OBJECTIVE: To investigate the role of microRNA-210 in the proliferation and odontogenic differentiation of rat dental pulp stem cells.
METHODS: The study was approved by the Ethics Committee of the Second Affiliated Hospital of Henan University of Chinese Medicine, China. The dental pulp tissues were collected from the mandibles of five Sprague-Dawley rats, and the pulp stem cells were isolated and identified. The dental pulp stem cells were divided into five groups. The normal control group was not treated. The cells in the miR-210 mimic negative control group were transfected with the miR-210 mimic negative control. The cells in the miR-210 mimic group were transfected with the miR-210 mimic. The cells in the miR-210 inhibitor negative control group were transfected with the miR-210 inhibitor negative control. The cells in the miR-210 inhibitor group were transfected with the miR-210 inhibitor negative control. Real-time quantitative PCR was used to detect the expression of microRNA-210, alkaline phosphatase, dentin sialophosphoprotein, osteopontin, osteocalcin, dentin matrix phosphoprotein-1 and GPCR-kinase interacting protein-2 in cells. Western blot was used to detect the expression of dentin sialophosphoprotein, osteopontin, osteocalcin and dentin matrix phosphoprotein-1 in cells. Cell counting kit-8 was used to detect cell proliferation. Cell alkaline phosphatase activity was detected by colorimetry. Alizarin red staining was used to detect the ability of mineral synthesis.
RESULTS AND CONCLUSION: (1) The proliferation activity of miR-210 mimic group was significantly higher than that of the other four groups (P < 0.01), while the proliferation activity of miR-210 inhibitor group was significantly lower than that of the other four groups (P < 0.01). (2) The activity of alkaline phosphatase in the miR-210 mimic group was significantly higher than that in the other four groups (P < 0.01), while the activity of alkaline phosphatase in the miR-210 inhibitor group was significantly lower than that in the other four groups (P < 0.01). (3) The results of alizarin red staining showed that the surfaces of cells in the miR-210 mimic group were covered with dark red nodules of different sizes owing to calcium salt deposition, and the number of dark red calcium nodules was higher than that in the other four groups. In the miR-210 inhibitor group, dark red calcium nodules were scattered on the cell surface, and the number of these nodules was lower than that in the other four groups. (4) The expressions of microRNA-210, alkaline phosphatase, dentin sialophosphoprotein, osteopontin, osteocalcin, dentin matrix phosphoprotein-1 and GPCR-kinase interacting protein-2 mRNAs and dentin sialophosphoprotein, osteopontin, osteocalcin and dentin matrix phosphoprotein-1 proteins were highest in the miR-210 mimic group and lowest in the miR-210 inhibitor group among the five groups, and there were significant differences among the five groups (P < 0.01). To conclude, microRNA-210 can promote the proliferation and odontogenic differentiation of rat dental pulp stem cells.

Key words: dental pulp stem cells, microRNA, microR-210, odontogenic differentiation, cell proliferation, alkaline phosphatase activity, mineral synthesis ability

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