骨形态发生蛋白-2、-4、-6、-7和-9差异性介导永生化成牙本质细胞成骨分化

doi:10.19439/j.sjos.2020.03.004

上海口腔医学 ›› 2020, Vol. 29 ›› Issue (3): 242-249.doi: 10.19439/j.sjos.2020.03.004

骨形态发生蛋白-2、-4、-6、-7和-9差异性介导永生化成牙本质细胞成骨分化

李静^1,2,3, 张艺^1,2,3, 王金华^1,2,3

1.重庆医科大学附属口腔医院儿童口腔科,重庆 400015;
2.口腔疾病与生物医学重庆市重点实验室,重庆 401120;
3.重庆市高校市级口腔生物医学工程重点实验室,重庆 401120

收稿日期:2020-02-20 修回日期:2020-03-24 出版日期:2020-06-25 发布日期:2020-07-29
通讯作者: 王金华,E-mail: 500190@hospital.cqmu.edu.cn
作者简介:李静(1989-),女,硕士,E-mail: 2017110892@stu.cqmu.edu.cn
基金资助:
重庆市基础研究和前沿技术研究计划（cstc2018jcyjAX0731）; 重庆市自然科学基金（cstc2018jcyjAX0731）; 2016年重庆高校创新团队建设计划资助项目（CXTDG201602006）

Bone morphogenetic protein-2, -4, -6, -7 and -9 differentially mediated osteogenic differentiation of immortalized odontoblasts

LI Jing^1,2,3, ZHANG Yi^1,2,3, WANG Jin-hua^1,2,3

1. Stomatological Hospital of Chongqing Medical University. Chongqing 400015;
2. Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences. Chongqing 401120;
3. Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education. Chongqing 401120, China

Received:2020-02-20 Revised:2020-03-24 Online:2020-06-25 Published:2020-07-29

摘要/Abstract

摘要： 目的研究永生化成牙本质细胞（immortalized odontoblasts,iODs）的间充质干细胞表型和骨形态发生蛋白（bone morphogenetic protein, BMP）-2、-4、-6、-7和-9差异介导iODs成骨分化的作用。方法使用SV40 T抗原永生化成牙本质细胞（odontoblasts）,建立iOD细胞株,并连续检测BMP的内源性表达。使用Ad-Easy技术合成表达BMP和GFP的重组腺病毒,使用MTT试剂盒检测iOD的增殖能力。通过免疫荧光检测iOD的间充质干细胞表面标志物。在体外,使用半定量RT-PCR、碱性磷酸酶活性、茜素红染色及油红O染色检测iOD的成骨和成脂分化能力。最后,使用micro-CT和组织学分析评估体内形成的异位矿化组织的体积和密度。采用SPSS 21.0软件包中的单因素方差分析和Scheffe的多重比较检验对结果进行分析。结果 SV40 T抗原有效地永生化OD。iOD细胞株保持良好的增殖活性,表达间充质干细胞表面标志物并具有多向分化能力。相比BMP-4、-6和-7,BMP-2和BMP-9具有更强的介导iOD的成骨分化作用。结论 ODs和成骨生长因子（如BMP-2和BMP-9）可用作骨组织生物工程的有效策略。

关键词: 骨形态发生蛋白, 成牙本质细胞, 永生化, 成骨分化

Abstract: PURPOSE: This study was aimed to investigate the mesenchymal stem cell (MSC) phenotypes of immortalized odontoblasts(iODs) and bone morphogenetic protein -2, -4, -6, -7, and -9 (BMPs) differentially regulate the mineralization of iODs. METHODS: ODs were immortalized by SV40 T antigen to establish iOD lineages, and the endogenous expression of BMPs was successively examined. Recombinant adenoviruses expressing BMPs and GFP were generated using Ad-Easy technology. The proliferation capability of iODs was examined using an MTT kit. MSC markers of iODs were examined by immunofluorescence. In vitro, semiquantitative RT-PCR, alkaline phosphatase(ALP) activity assay, matrix mineralization assay and oil red O staining assay were used to examine the osteogenic and adipogenic differentiation capabilities of iODs. Statistical significance among groups was analyzed by one-way analysis of variance and Scheffe's multiple comparison test was SPSS 21.0 software package. Finally, the volume and density of ectopic mineralized tissues formed in vivo were assessed by micro-CT and histological analysis. RESULTS: ODs can be efficiently immortalized by SV40 T antigen, and the resulting iODs maintained an excellent proliferative activity, expressed certain MSC markers and possessed multiple differentiation capabilities. BMP-2 and BMP-9 regulated iODs osteogenic differentiation better than BMP-4, -6, and -7. CONCLUSIONS: Our findings suggest that ODs and osteogenic growth factors such as BMP-2 and BMP-9 can be used as an efficacious strategy for bone tissue engineering.

Key words: Bone morphogenetic protein, Odontoblasts, Immortalized odontoblasts, Osteogenic differentiation

中图分类号:

R781.3

李静, 张艺, 王金华. 骨形态发生蛋白-2、-4、-6、-7和-9差异性介导永生化成牙本质细胞成骨分化[J]. 上海口腔医学, 2020, 29(3): 242-249.

LI Jing, ZHANG Yi, WANG Jin-hua. Bone morphogenetic protein-2, -4, -6, -7 and -9 differentially mediated osteogenic differentiation of immortalized odontoblasts[J]. Shanghai Journal of Stomatology, 2020, 29(3): 242-249.

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骨形态发生蛋白-2、-4、-6、-7和-9差异性介导永生化成牙本质细胞成骨分化

Bone morphogenetic protein-2, -4, -6, -7 and -9 differentially mediated osteogenic differentiation of immortalized odontoblasts

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