华西口腔医学杂志 ›› 2018, Vol. 36 ›› Issue (4): 378-383.doi: 10.7518/hxkq.2018.04.006

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神经肽P物质及骨形态发生蛋白信号通路在ST2细胞成骨分化过程中的作用

惠婷(), 张广灿, 冯丹丹, 汲平()   

  1. 山东大学口腔医院修复科 山东省口腔组织再生重点实验室,济南 250012
  • 收稿日期:2017-12-16 修回日期:2018-04-02 出版日期:2018-08-01 发布日期:2018-08-01
  • 作者简介:

    惠婷,住院医师,硕士,E-mail:545638123@qq.com

Role of neuropeptide substance P and the bone morphogenetic protein signaling pathway in osteogenic differentiation of ST2 cells

Ting Hui(), Guangcan Zhang, Dandan Feng, Ping Ji()   

  1. Dept. of Prosthodontics, Hospital of Stomatology, Shandong University, Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Jinan 250012, China
  • Received:2017-12-16 Revised:2018-04-02 Online:2018-08-01 Published:2018-08-01

摘要:

目的 探讨神经肽P物质(SP)在ST2细胞(小鼠骨髓间充质干细胞)成骨分化过程中的作用及机制,以期为颞下颌关节骨关节炎的治疗提供依据。方法 对第3代ST2细胞分别用0、10-10、10-8 、10-6、10-5 mol·L-1 SP培养,24、48、72 h后采用CCK-8实验检测细胞增殖情况。以10-6 mol·L-1 SP培养第3代ST2细胞1、3、5、7 d后,采用酶联免疫吸附试验(ELISA)检测上清液中碱性磷酸酶(ALP)、Ⅰ型胶原蛋白(CollaⅠ)和骨钙素(OCN)的表达,免疫荧光染色检测细胞ALP活性。分别用SP、Noggin(骨形态发生蛋白信号通路抑制剂)、SP+Noggin和2%胎牛血清培养ST2细胞,采用ELISA法检测上清液中ALP、CollaⅠ和OCN的表达。结果 CCK-8结果显示,24、48、72 h时均以10-6 mol·L-1 SP促进ST2细胞增殖活性最为明显(P<0.01)。ELISA结果显示,ALP表达在5 d时较对照组差异最为明显(P<0.01),CollaⅠ和OCN的表达在7 d时较对照组差异最为明显(P<0.05);免疫荧光结果显示,ALP活性在5 d时最强;加入抑制剂Noggin后,ALP、CollaⅠ和OCN表达量均降低。结论 SP可促进ST2细胞增殖和成骨分化,骨形态发生蛋白信号通路可能参与了此过程。

关键词: 神经肽P物质, 骨髓间充质干细胞, 骨形态发生蛋白信号通路, 成骨分化

Abstract:

Objective This study aimed to investigate the role and mechanism of neuropeptide substance P (SP) in ST2 cell (bone mesenchymal stem cells of mice) osteogenic differentiation to provide a basis for the treatment of temporomandibular joint osteoarthritis. Methods Third-generation ST2 cells were cultured with different concentrations of SP (0, 10-10, 10-8, 10-6, and 10-5 mol·L-1). After 24, 48, and 72 h, cell proliferation was detected by CCK-8. The ST2 cells were cultured with 10-6 mol·L-1 SP for 1, 3, 5, and 7 days. Subsequently, the expression of alkaline phosphatase (ALP), collagen typeⅠ(CollaⅠ), and osteocalcin (OCN) in the culture supernatant was tested by enzyme-linked immunosorbent assay (ELISA). ALP activity was detected by immunofluorescence staining. The ST2 cells were cultured with SP, Noggin (inhibitor of the bone morphogenetic protein signaling pathway), SP+Noggin, and 2% fetal bovine serum, respectively. Finally, the expression of ALP, CollaⅠ, and OCN in the culture supernatant was tested by ELISA. Results CCK-8 showed that the effect of cell proli-feration was most obvious when the SP concentration was 10-6 mol·L-1 (P<0.01). The ELISA results demonstrated that ALP expression significantly increased at day 5 compared with that in the control group (P<0.01), whereas the expression of CollaⅠand OCN significantly increased at day 7 (P<0.05). Immunofluorescence results showed that ALP activity was strongest at day 5. The expression of ALP, CollaⅠ, and OCN decreased after Noggin addition (P<0.05). Conclusion SP can promote the proliferation and osteogenic differentiation of ST2 cells, and the bone morphogenetic protein signaling pathway may be involved in this process.

Key words: neuropeptide substance P, bone mesenchy-mal stem cells, bone morphogenetic protein signaling pathway, osteogenic differentiation

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