小分子鹰嘴豆芽素A对人牙髓干细胞成骨分化的影响

doi:10.19438/j.cjoms.2021.03.004

中国口腔颌面外科杂志 ›› 2021, Vol. 19 ›› Issue (3): 205-212.doi: 10.19438/j.cjoms.2021.03.004

小分子鹰嘴豆芽素A对人牙髓干细胞成骨分化的影响

谢昕儒^1,*, 冉雪会^2,*, 徐伟峰¹, 刘秀明¹, 张翔凯³, 曹玲燕^4#, 张善勇^1#

1.上海交通大学医学院附属第九人民医院口腔外科,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,上海 200011;
3.上海交通大学医学院附属第九人民医院口腔颌面-头颈肿瘤科,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;
4.上海交通大学医学院附属第九人民医院口腔修复科,国家口腔疾病临床医学研究中心, 上海口腔医学先进技术与材料工程技术研究中心,上海市口腔医学重点实验室,上海 200011

收稿日期:2020-11-27 修回日期:2021-01-15 发布日期:2021-07-16
通讯作者: 曹玲燕,E-mail：cly_linya@163.com;张善勇,E-mail:zhangshanyong@126.com。^#共同通信作者
作者简介:谢昕儒（1994-）,女,硕士研究生,E-mail：xxru1112@foxmail.com;冉雪会（2000-）,女,在读本科,E-mail：2764608934@qq.com。^*并列第一作者

Effects of small molecule biochanin A on osteogenic differentiation of human dental pulp stem cells

XIE Xin-ru¹, RAN Xue-hui², XU Wei-feng¹, LIU Xiu-ming¹, ZHANG Xiang-kai³, CAO Ling-yan³, ZHANG Shan-yong¹

1. Department of Oral 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;
2. Shanghai Jiao Tong University School of Medicine. Shanghai 200011;
3. Department of Oromaxillofacial Head and Neck Oncology, 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;
4. Department of Prothodontics, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; National Clinical Research Center for Oral Diseases; Shanghai Engineering Research Center of Advanced Dental Technology and Materials; Shanghai Key Laboratory of Stomatology. Shanghai 200011, China

Received:2020-11-27 Revised:2021-01-15 Published:2021-07-16

摘要/Abstract

摘要： 目的探讨不同浓度鹰嘴豆芽素A（biochanin A,BCA）对人牙髓干细胞（human dental pulp stem cells,hDPSCs）成骨分化的影响及相关分子机制。方法通过组织块法分离培养原代人牙髓干细胞,流式细胞术鉴定其细胞表型。通过CCK-8法检测不同浓度BCA对hDPSCs增殖活性的影响,通过q-PCR、碱性磷酸酶染色、茜素红染色等分析0、1、5、10、20、30 μmol/L BCA对hDPSCs成骨分化的作用,通过蛋白免疫印迹研究BCA对hDPSCs成骨分化过程中MAPK信号通路的影响。采用SPSS 16.0软件包对数据进行统计学分析。结果实验获取的hDPSCs经流式细胞术鉴定,符合间充质干细胞特征。CCK-8结果显示,25 μmol/L以下浓度的BCA对hDPSCs增殖无影响,30 μmol/L以上抑制hDPSCs增殖。q-PCR结果显示,10 μmol/L BCA显著提升hDPSCs成骨分化过程中标志基因ALP、COL-1、OPN、OCN的表达水平;碱性磷酸酶及茜素红染色显示,10 μmol/L BCA组比对照组及其他浓度组染色明显增强;蛋白免疫印迹实验显示,BCA激活P38/MAPK信号通路,但对ERK及JNK无明显激活作用。结论组织块法可获取纯度较高的hDPSCs,BCA可能通过激活P38/MAPK信号通路,从而促进hDPSCs成骨向分化。

关键词: 鹰嘴豆芽素A, 人牙髓干细胞, 成骨分化, MAPK信号通路

Abstract: PURPOSE: To study the effects of different concentrations of biochanin A (BCA) on osteogenic differentiation of human dental pulp stem cells (hDPSCs) and its related molecular mechanisms. METHODS: Primary human dental pulp stem cells were isolated and cultured by tissue explant method, cell phenotypes were identified by flow cytometry. Effects of different concentrations of BCA on proliferation of hDPSCs were detected by CCK-8 method, and effects of different concentrations of BCA on osteogenic differentiation of hDPSCs were analyzed by q-PCR, alkaline phosphatase (ALP) staining, and alizarin red staining. Finally, the influence of BCA on MAPK signaling pathway during osteogenic differentiation of hDPSCs was investigated through Western blot. SPSS 16.0 software package was used for statistical analysis of the data. RESULTS: hDPSCs obtained in the experiment were conformed to have the characteristics of mesenchymal stem cells by flow cytometry. CCK-8 results showed that BCA below 25 μmol/L had no effect on cell activity of hDPSCs, while over 30 μmol/L BCA inhibited the proliferation of hDPSCs. 10 μmol/L BCA significantly increased the expression of osteogenic-related genes ALP, COL-1, OPN and OCN. The staining of ALP and alizarin red was also more obvious than that of the control group and the other concentration groups. Western blot displayed that BCA activated P38/MAPK signaling pathway, but had no obvious effect on ERK and JNK pathway. CONCLUSIONS: Tissue explant method can obtain hDPSCs with higher purity. BCA may promote osteogenic differentiation of hDPSCs by activating P38/MAPK signaling pathway.

Key words: Biochanin A, Human dental pulp stem cells, Osteogenic differentiation, MAPK signaling pathway

中图分类号:

Q254

谢昕儒, 冉雪会, 徐伟峰, 刘秀明, 张翔凯, 曹玲燕, 张善勇. 小分子鹰嘴豆芽素A对人牙髓干细胞成骨分化的影响[J]. 中国口腔颌面外科杂志, 2021, 19(3): 205-212.

XIE Xin-ru, RAN Xue-hui, XU Wei-feng, LIU Xiu-ming, ZHANG Xiang-kai, CAO Ling-yan, ZHANG Shan-yong. Effects of small molecule biochanin A on osteogenic differentiation of human dental pulp stem cells[J]. China J Oral Maxillofac Surg, 2021, 19(3): 205-212.

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小分子鹰嘴豆芽素A对人牙髓干细胞成骨分化的影响

Effects of small molecule biochanin A on osteogenic differentiation of human dental pulp stem cells

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