p38 MAPK通路调控BMP9诱导hPDLSCs成骨分化的体外研究

doi:10.19439/j.sjos.2018.06.007

上海口腔医学 ›› 2018, Vol. 27 ›› Issue (6): 596-601.doi: 10.19439/j.sjos.2018.06.007

p38 MAPK通路调控BMP9诱导hPDLSCs成骨分化的体外研究

张奕, 胡婷, 叶国, 向学熔, 胡娜

重庆医科大学附属口腔医院牙周病科,口腔疾病与生物医学重庆市重点实验室, 重庆市高校市级口腔生物医学工程重点实验室,重庆 401147

收稿日期:2017-11-14 出版日期:2018-12-25 发布日期:2019-01-11
通讯作者: 向学熔,E-mail:cqzxfzp@sina.com
作者简介:张奕（1993-）,女,硕士,E-mail:2534237069@qq.com

p38 MAPK pathway mediated BMP9-induced osteogenetic differentiation of hPDLSCs

ZHANG Yi, HU Ting, YE Guo, XIANG Xue-rong, HU Na

Department of Periodontology, Stomatological Hospital of Chongqing Medical University; Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences; Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education. Chongqing 401147, China

Received:2017-11-14 Online:2018-12-25 Published:2019-01-11
Supported by:
重庆市科卫联合医学科研项目（2018ZDXM031）; 重庆市卫生计生委医学科研项目(2015MSXM048); 重庆高校创新团队建设计划资助项目（CXTDG201602006）; 重庆市高校市级口腔生物医学工程重点实验室资助项目

摘要/Abstract

摘要： 目的: 检测骨形成发生蛋白9（bone morphogenetic proteins 9,BMP9）对人牙周膜干细胞（human periodontal ligament stem cells,hPDLSCs ）的成骨诱导分化能力,并探讨p38 MAPK通路在该成骨分化中的作用。方法: 培养hPDLSCs,腺病毒载体（Ad-BMP9)感染hPDLSCs后,通过碱性磷酸酶(alkaline phosphatase, ALP)活性测定与染色、定量聚合酶链反应(quantitative polymerase chain reaction, qPCR)、钙盐沉积等方法,分别检测ALP、骨桥蛋白(osteopontin, OPN)、骨钙素(osteocalcin, OCN)的表达及钙盐沉积量,评价BMP9诱骨分化的能力。在Ad-BMP9感染hPDLSCs 36 h 后,检测BMP9作用hPDLSCs后对p38及MKK3/6磷酸化（p-p38, p-MKK3/6)的影响,以及p38 MAPK通路抑制剂SB203580预处理后BMP9-p38-MAPK通路对hPDLSCs 成骨分化的影响。采用SPSS16.0软件包对数据进行统计学分析。结果: 在Ad-BMP9作用下,ALP活性、OPN和 OCN的表达均显著高于对照组（P<0.01), ALP染色与钙盐沉积结果与之吻合。Western印迹法检测发现,BMP9可增强p-p38、p-MKK3/6的表达。加入p38 MAPK通路抑制剂后,ALP、及OPN、OCN的表达均显著降低（P<0.01）,钙盐沉积、基质矿化也显著减弱。结论: 在hPDLSCs的分化过程中,BMP9对其具有诱骨分化能力。MKK3/6-p38 MAPK通路参与了该成骨分化过程且具有正向调节作用。

关键词: 人牙周膜干细胞, BMP9, 成骨分化, p38 MAPK

Abstract: PURPOSE: To determine the effect of p38 MAPK signaling pathway on BMP9-induced osteogenetic differentiation of human periodontal ligament stem cells( hPDLSCs). METHODS: hPDLSCs were collected in vitro, and adenovirus vectors were used to infect hPDLSCs; then the activity and staining of alkaline phosphatase(ALP) were detected ,the expression of osteopontin (OPN) and osteocalcin(OCN) were detected by qPCR ,and the calcium nodule deposition was used to analyse the ability of BMP9-induced hPDLSCs osteogenetic differentiation. Phosphorylation of p38 and MKK3/6 was detected after hPDLSCs was intervened with Ad-BMP9 and SB203580 ( inhibitor of p38 MAPK signaling pathway) for 36 h respectively for the effect of the signaling pathway on osteogenic differentiation. SPSS16.0 software package was used for statistical analysis. RESULTS: Under the action of Ad-BMP9, the activity of ALP, the levels of osteopontin and osteocalcin genes were significantly higher than the control group (P<0.01). Staining of ALP and the calcium nodule deposition were consistent with the activity of ALP, the levels of osteopontin and osteocalcin. Western blot demonstrated that the expression of p-p38 and p-MKK3/6 was increased significantly. After adding SB203580, the expression of ALP, OPN and OCN was decreased significantly (P<0.01),and the calcium mineral deposits were also decreased. CONCLUSIONS: During hPDLSCs differentiation, BMP9 can induce osteogenesis, and MKK3/6-p38-MAPK pathway was involved in the osteogenesis and had positive regulation for osteogenesis of hPDLSCs.

Key words: Human periodontal ligament stem cells, Bone morphogenetic protein 9, Osteogenesis differentiation, p38 MAPK

中图分类号:

R781.4

张奕, 胡婷, 叶国, 向学熔, 胡娜. p38 MAPK通路调控BMP9诱导hPDLSCs成骨分化的体外研究[J]. 上海口腔医学, 2018, 27(6): 596-601.

ZHANG Yi, HU Ting, YE Guo, XIANG Xue-rong, HU Na. p38 MAPK pathway mediated BMP9-induced osteogenetic differentiation of hPDLSCs[J]. Shanghai Journal of Stomatology, 2018, 27(6): 596-601.

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p38 MAPK通路调控BMP9诱导hPDLSCs成骨分化的体外研究

p38 MAPK pathway mediated BMP9-induced osteogenetic differentiation of hPDLSCs

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