Department of Stomatology, Jinling Hospital, Jinling Medical School of Nanjing University, Medical School of Southern Medical University, Clinic School of Nanjing Medical University, Clinic School of the Fourth Military Medical University, Nanjing, Jiangsu 210002, People's Republic of China; State Key laboratory of Military Stomatology, Department of Prosthodontics, School of Stomatology, The Fourth Military Medical University, Xian, Shaanxi 710023, People's Republic of China.
Department of Stomatology, Jinling Hospital, Jinling Medical School of Nanjing University, Medical School of Southern Medical University, Clinic School of Nanjing Medical University, Clinic School of the Fourth Military Medical University, Nanjing, Jiangsu 210002, People's Republic of China.
Life Sci. 2018 Jun 1;202:175-181. doi: 10.1016/j.lfs.2018.03.026. Epub 2018 Mar 16.
in vitro effects of bone morphogenetic protein 7 (BMP-7) on proliferation and differentiation of dental pulp stem cells (DPSCs) have not been investigated, nor has an appropriate dose been established.
Human DPSCs obtained from healthy volunteers were cultured with BMP-7 at 25, 50, and 100 ng/ml. Cell viability was measured by Cell Counting Kit-8 assay. Expression profiles of selected odontogenic differentiation-related markers in DPSCs were evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunocytochemistry, and western blot analysis. Mineralization of DPSCs was evaluated by alizarin red staining. The Smad5 signaling pathway was examined by qRT-PCR and western blot analysis.
Diminished cell viability was found in DPSCs induced with 25, 50, and 100 ng/ml of BMP-7 for 7 days, showing a dose-response effect (P-trend = 0.03). DSPP, OCN, DMP-1, and RUNX2 were upregulated by BMP-7 induction after 7 and 14 days, especially at 50 and 100 ng/ml (P < 0.05). Immunocytochemical staining revealed strong expression of DSPP, DMP-1 and ALP in DPSCs induced by BMP-7, whereas null or weak expression in untreated cells. Western blot analysis confirmed over-expression of DSPP in cells induced by BMP-7. Alizarin red staining confirmed formation of mineralized nodules 4 weeks after BMP-7 induction. BMP-7 treated cells showed dose-dependently increased expression of BMPR1A, Smad5, and p-Smad5.
Our data indicated that BMP-7 at 50 ng/ml and 100 ng/ml was capable to induce DPSCs toward odontogenic differentiation through the Smad5 signaling pathway and not dramatically halt cell proliferation in vitro.
骨形成蛋白 7（BMP-7）对牙髓干细胞（DPSCs）增殖和分化的体外影响尚未得到研究，也未确定合适的剂量。
从健康志愿者中分离培养人牙髓干细胞，用 BMP-7 以 25、50 和 100ng/ml 处理细胞。通过细胞计数试剂盒-8 法测量细胞活力。采用定量逆转录聚合酶链反应（qRT-PCR）、免疫细胞化学和 Western blot 分析检测 DPSCs 中选定的牙源性分化相关标志物的表达谱。通过茜素红染色评估 DPSCs 的矿化情况。通过 qRT-PCR 和 Western blot 分析检测 Smad5 信号通路。
用 25、50 和 100ng/ml 的 BMP-7 诱导 DPSCs 7 天后，发现细胞活力降低，呈剂量反应效应（P 趋势=0.03）。BMP-7 诱导 7 和 14 天后，DSPP、OCN、DMP-1 和 RUNX2 上调，尤其是在 50 和 100ng/ml 时（P<0.05）。免疫细胞化学染色显示，BMP-7 诱导的 DPSCs 中 DSPP、DMP-1 和 ALP 表达强烈，而未经处理的细胞则呈阴性或弱阳性。Western blot 分析证实，BMP-7 诱导的细胞中 DSPP 过表达。BMP-7 诱导 4 周后，茜素红染色证实形成矿化结节。BMP-7 处理的细胞表现出 BMPR1A、Smad5 和 p-Smad5 的剂量依赖性表达增加。
我们的数据表明，BMP-7 在 50ng/ml 和 100ng/ml 时能够通过 Smad5 信号通路诱导 DPSCs 向牙源性分化，而不会显著抑制体外细胞增殖。
