尼古丁通过调控Toll样受体4抑制牙周膜干细胞的成骨分化能力

doi:10.7518/hxkq.2017.04.005

华西口腔医学杂志 ›› 2017, Vol. 35 ›› Issue (4): 368-372.doi: 10.7518/hxkq.2017.04.005

尼古丁通过调控Toll样受体4抑制牙周膜干细胞的成骨分化能力

闫娈(), 杨德琴()

重庆医科大学附属口腔医院牙体牙髓病科,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:2017-01-06 修回日期:2017-04-02 出版日期:2017-08-01 发布日期:2017-08-01
作者简介:
闫娈,住院医师,硕士,E-mail:24425122@qq.com
基金资助:
国家自然科学基金面上项目（31571508,31371473）;重庆市第七批重点学科建设项目（牙体牙髓病学,2011年）;重庆市卫生局医学科研计划重点项目（2011-1-062）;重庆市渝北区科技项目[渝北财教(2011)33号]

The effect of Toll-like receptor 4 in nicotine suppressing the osteogenic potential of periodontal ligament stem cells

Luan Yan(), Deqin Yang()

Dept. of Conservative Dentistry and Endodontics, 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:2017-01-06 Revised:2017-04-02 Online:2017-08-01 Published:2017-08-01
Supported by:
National Natural Science Foundation of China (31571508, 31371473);The Seventh Batch of Key Discipline Construction Project of Chongqing—Endodontics and Operative Dentistry (2011);Key Project of Medical Research Program of Chongqing Municipal Health Bureau (2011-1-062);Science and Technology Project of Yubei District in Chongqing [Yubei Finance and Education (2011) No.33]

摘要/Abstract

摘要：

目的探讨尼古丁对牙周膜干细胞（PDLSCs）增殖和成骨分化能力的调控作用,检测尼古丁能否通过调控Toll样受体4（TLR4）抑制PDLSCs的增殖及成骨分化能力。方法培养PDLSCs,采用流式细胞仪检测PDLSCs表面抗原标志,WST-1试剂盒检测不同浓度尼古丁刺激后PDLSCs增殖能力的改变。采用茜素红染色观察PDLSCs经不同浓度尼古丁刺激和成骨诱导后的矿化结节生产情况。运用实时定量聚合酶链反应（RT-PCR）和Western blot检测经尼古丁刺激后,PDLSCs成骨能力相关基因和蛋白的改变,以及尼古丁联合TLR4抑制剂TAK-242刺激后,PDLSCs成骨能力相关基因及蛋白的改变。结果培养的细胞表达间充质干细胞表面标志物CD90和CD105,证实为PDLSCs。与对照组相比,培养3 d后,尼古丁浓度为10^-4 mol·L^-1的PDLSCs的增殖能力受到明显抑制（P<0.05）;成骨诱导21 d后,10^-4 mol·L^-1尼古丁刺激组茜素红染色矿化结节明显减少。RT-PCR反应及Western blot显示：与对照组相比,尼古丁刺激组PDLSCs的碱性磷酸酶、骨钙素、Runt相关转录因子2的基因及蛋白表达量均降低（P<0.05）,加入TLR4抑制剂TAK-242后,尼古丁的抑制效应减弱。结论尼古丁可能通过TLR4信号通路抑制PDLSCs的增殖及成骨分化能力。

关键词: 尼古丁, 牙周膜干细胞, Toll样受体4, 成骨分化

Abstract:

Objective To explore the impact of nicotine on proliferation and osteogenic capability of periodontal ligament stem cells (PDLSCs), and the role of Toll-like receptor 4 (TLR4) in nicotine, suppressing the osteogenic capability of PDLSCs. Methods PDLSCs were cultured in vitro, and the flow cytometer was used to identify the surface antigen markers of PDLSCs. WST-1 was used to detect the proliferation ability of PDLSCs, which were stimulated by different concentrations of nicotine. Alizarin red staining was used to observe the formation of mineralized nodules after PDLSCs stimulation with different con-centrations of nicotine. Real-time polymerase chain reaction (RT-PCR) and Western blot were used to detect the change in osteogenic potential of PDLSCs stimulated by nicotine, after TAK-242, and with the inhibitor of TLR4. Results PDLSCs expressed mesenchymal stem cell-associated mar-kers CD90 and CD105. When the concentration of nicotine was 10^-4mol·L^-1, the PDLSC proliferation could be suppressed after 3 d compared with the control group (P<0.05). The amount of mineralized nodules reduced after osteogenic differentiation at 21 d by alizarin red staining. RT-PCR and Western blot showed the expression levels of alkaline phosphatase (ALP), and osteocalcin (OCN), and the Runt-related transcription factor-2 (Runx-2) were lower than in the control group when nicotine suppressed the PDLSCs (P<0.05). This effect was atte-nuated after TAK-242 was added. Conclusion Nicotine suppresses the proliferation and osteogenic capability of PDLSCs, which may be regulated by TLR4.

Key words: nicotine, periodontal ligament stem cells, Toll-like receptors 4, osteogenic differentiation

中图分类号:

R781.4

闫娈, 杨德琴. 尼古丁通过调控Toll样受体4抑制牙周膜干细胞的成骨分化能力[J]. 华西口腔医学杂志, 2017, 35(4): 368-372.

Luan Yan, Deqin Yang. The effect of Toll-like receptor 4 in nicotine suppressing the osteogenic potential of periodontal ligament stem cells[J]. West China Journal of Stomatology, 2017, 35(4): 368-372.

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组别	1 d	2 d	3 d	4 d	5 d
10^-4 mol·L^-1	0.37±0.27	0.37±0.71	0.37±0.02*	0.20±0.02*	0.17±0.01*
10^-5 mol·L^-1	0.35±0.01	0.43±0.03	1.01±0.07	1.25±0.06	1.50±0.05*
10^-6 mol·L^-1	0.34±0.01	0.57±0.02	1.38±0.03	1.78±0.03	2.13±0.12
对照组	0.34±0.01	0.48±0.01	1.29±0.02	1.97±0.03	2.25±0.06

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尼古丁通过调控Toll样受体4抑制牙周膜干细胞的成骨分化能力

The effect of Toll-like receptor 4 in nicotine suppressing the osteogenic potential of periodontal ligament stem cells

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