State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine of Ministry of Education (KLOBM), School and Hospital of Stomatology, Wuhan University, Wuhan, China, 430079.
J Endod. 2011 Jul;37(7):948-54. doi: 10.1016/j.joen.2011.03.030. Epub 2011 May 13.
Krüppel-like factor 4 (KLF4) plays an important role in cytodifferentiation and proliferation. Our previous study showed that KLF4 was specifically expressed in polarizing and elongating odontoblasts. However, the role of KLF4 in odontoblast differentiation was still unknown. The purpose of this study was to investigate the role of KLF4 in odontoblastic differentiation of human dental pulp cells (hDPCs).
hDPCs were treated with odontoblastic induction medium. Odontoblastic differentiation was determined by the detection of alkaline phosphatase (ALPase) activity and the expression of mineralization-related genes including ALP, dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1). Also, cell proliferation ability was examined by the 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Simultaneously, messenger RNA and protein levels of KLF4 were detected. pKLF4-IRES2-EGFP plasmid encoding full-length KLF4 was constructed to overexpress KLF4, and biologic effects of KLF4 on hDPCs were investigated by the evaluation of ALPase activity and the detection of ALP, DSPP, and DMP-1 expression and analysis of cell proliferation ability.
ALPase activity and the expression of odontoblastic differentiation markers progressively increased in hDPCs cultured with odontoblastic induction medium. Meanwhile, the proliferation ability decreased in this procedure; messenger RNA and protein levels of KLF4 increased significantly on day 5 after the odontoblastic induction of hDPCs and kept increasing until day 14. hDPCs showed up-regulated activity of ALPase and the expression of mineralization-related genes, including ALP, DMP-1, and dentin sialoprotein (DSP), after KLF4 overexpression. Besides, the proliferation ability of hDPCs decreased significantly in the KLF4 overexpression group by EdU incorporation assay.
Our findings suggest that KLF4 is able to promote odontoblastic differentiation of hDPCs and inhibit proliferation of hDPCs.
Krüppel 样因子 4（KLF4）在细胞分化和增殖中发挥重要作用。我们之前的研究表明，KLF4 特异性表达于极化和伸长的成牙本质细胞中。然而，KLF4 在成牙本质细胞分化中的作用尚不清楚。本研究旨在探讨 KLF4 在人牙髓细胞（hDPCs）成牙本质分化中的作用。
用成牙本质诱导培养基处理 hDPCs。通过碱性磷酸酶（ALPase）活性检测和矿化相关基因（包括 ALP、牙本质涎磷蛋白（DSPP）和牙本质基质蛋白-1（DMP-1）的表达）的检测来确定成牙本质分化。同时，通过 5-乙炔基-2'-脱氧尿苷（EdU）掺入试验检测细胞增殖能力。构建了编码全长 KLF4 的 pKLF4-IRES2-EGFP 质粒以过表达 KLF4，并通过 ALPase 活性评估和 ALP、DSPP 和 DMP-1 表达的检测以及分析细胞增殖能力来研究 KLF4 对 hDPCs 的生物学效应。
在成牙本质诱导培养基中培养的 hDPCs 中，ALPase 活性和牙本质分化标志物的表达逐渐增加，同时细胞增殖能力下降。在 hDPCs 成牙本质诱导的第 5 天，KLF4 的信使 RNA 和蛋白水平显著增加，并持续增加到第 14 天。hDPCs 过表达 KLF4 后，ALPase 活性和矿化相关基因（包括 ALP、DMP-1 和牙本质涎蛋白（DSP））的表达上调。此外，EdU 掺入试验显示 KLF4 过表达组 hDPCs 的增殖能力显著下降。
我们的研究结果表明，KLF4 能够促进 hDPCs 的成牙本质分化并抑制 hDPCs 的增殖。
