Department of Orthodontics, Hospital of Stomatology, Zhejiang University, Hangzhou, Zhejiang, China.
PLoS One. 2013 Jul 29;8(7):e69967. doi: 10.1371/journal.pone.0069967. Print 2013.
Multipotent human dental follicle cells (HDFCs) have been intensively studied in periodontal regeneration research, yet the role of Notch1 in HDFCs has not been fully understood. The aim of the current study is to explore the role of Notch1 signaling in HDFCs self-renewal and proliferation. HDFCs were obtained from the extracted wisdom teeth from adolescent patients. Regulation of Notch1 signaling in the HDFCs was achieved by overexpressing the exogenous intracellular domain of Notch1 (ICN1) or silencing Notch1 by shRNA. The regulatory effects of Notch1 on HDFC proliferation, cell cycle distribution and the expression of cell cycle regulators were investigated through various molecular technologies, including plasmid construction, retrovirus preparation and infection, qRT-PCR, western blot, RBP-Jk luciferase reporter and cell proliferation assay. Our data clearly show that constitutively activation of Notch1 stimulates the HDFCs proliferation while inhibition of the Notch1 suppresses their proliferation in vitro. In addition, the HDFCs proliferation is associated with the increased expression of cell cycle regulators, e.g. cyclin D1, cyclin D2, cyclin D3, cyclin E1, CDK2, CDK4, CDK6, and SKP2 and the decreased expression of p27 (kip1). Moreover, our data show that the G1/S phase transition (indicating proliferation) and telomerase activity (indicating self-renewal) can be enhanced by overexpression of ICN1 but halted by inhibition of Notch1. Together, the current study provides evidence for the first time that Notch1 signaling regulates the proliferation and self-renewal capacity of HDFCs through modulation of the G1/S phase transition and the telomerase activity.
多能人牙囊细胞（HDFCs）在牙周再生研究中受到了广泛的研究，但 Notch1 在 HDFCs 中的作用尚未完全了解。本研究旨在探讨 Notch1 信号通路在 HDFCs 自我更新和增殖中的作用。HDFCs 从青少年患者拔出的智齿中获得。通过过表达 Notch1 的外源性细胞内结构域（ICN1）或通过 shRNA 沉默 Notch1 来调节 HDFCs 中的 Notch1 信号通路。通过质粒构建、逆转录病毒制备和感染、qRT-PCR、western blot、RBP-Jk 荧光素酶报告基因和细胞增殖测定等各种分子技术，研究了 Notch1 对 HDFC 增殖、细胞周期分布和细胞周期调控因子表达的调节作用。我们的数据清楚地表明，Notch1 的组成性激活刺激 HDFCs 的增殖，而 Notch1 的抑制则抑制其在体外的增殖。此外，HDFCs 的增殖与细胞周期调控因子的表达增加有关，例如 cyclin D1、cyclin D2、cyclin D3、cyclin E1、CDK2、CDK4、CDK6 和 SKP2，以及 p27（kip1）的表达减少。此外，我们的数据表明，通过 ICN1 的过表达可以增强 G1/S 期转变（表示增殖）和端粒酶活性（表示自我更新），而 Notch1 的抑制则阻止了 G1/S 期转变和端粒酶活性。总之，本研究首次提供了证据，表明 Notch1 信号通路通过调节 G1/S 期转变和端粒酶活性来调节 HDFCs 的增殖和自我更新能力。
