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.
Hua Xi Kou Qiang Yi Xue Za Zhi. 2017 Aug 1;35(4):368-372. doi: 10.7518/hxkq.2017.04.005.
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 concentrations 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 markers CD90 and CD105. When the concentration of nicotine was 10⁻⁴ mol·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 attenuated after TAK-242 was added. Conclusion Nicotine suppresses the proliferation and osteogenic capability of PDLSCs, which may be regulated by TLR4.
目的 探讨尼古丁对牙周膜干细胞（PDLSCs）增殖和成骨能力的影响，以及Toll样受体4（TLR4）在尼古丁抑制PDLSCs成骨能力中的作用。方法 体外培养PDLSCs，采用流式细胞仪鉴定PDLSCs的表面抗原标志物。用WST-1检测不同浓度尼古丁刺激后PDLSCs的增殖能力。用茜素红染色观察不同浓度尼古丁刺激PDLSCs后矿化结节的形成。采用实时聚合酶链反应（RT-PCR）和蛋白质印迹法检测TAK-242及TLR4抑制剂作用后尼古丁刺激的PDLSCs成骨潜能的变化。结果 PDLSCs表达间充质干细胞相关标志物CD90和CD105。当尼古丁浓度为10⁻⁴ mol·L⁻¹时，与对照组相比，3 d后PDLSCs增殖受到抑制（P<0.05）。茜素红染色显示，成骨分化21 d后矿化结节数量减少。RT-PCR和蛋白质印迹法显示，尼古丁抑制PDLSCs时，碱性磷酸酶（ALP）、骨钙素（OCN）和 runt相关转录因子2（Runx-2）的表达水平低于对照组（P<0.05）。加入TAK-242后，这种作用减弱。结论 尼古丁抑制PDLSCs的增殖和成骨能力，这可能受TLR4调控。
