Graduate School at Shenzhen, Tsinghua University, Shenzhen, China.
Department of Chemistry, Tsinghua University, Beijing, China.
Int Endod J. 2017 Dec;50 Suppl 2:e52-e62. doi: 10.1111/iej.12811. Epub 2017 Aug 20.
To determine the roles of autophagy and Notch signalling in mineral trioxide aggregate (MTA)-mediated proliferation and differentiation of human dental pulp cells (hDPCs).
hDPCs were separated from human healthy molar teeth using collagenase I/dispase II digestion and then cultured in α-MEM medium with 15% foetal bovine serum. hDPCs were seeded in 96-well plates, and cell counting kit assays were carried out to test their viability. Real-time quantitative polymerase chain reaction (qPCR) was used to evaluate ALP, Runx2, Notch1, Hes1 and Hey1 mRNA levels. Notch1, hes1, LC3 and p62 protein levels were quantified by Western blotting. Colocalization of LC3 and Notch1 was measured by immunofluorescence. Two-tailed Student's t-tests were used for statistical analysis.
Autophagic flux was significantly (P < 0.05) inhibited by MTA extracts, causing Notch degradation arrest. This resulted in the promotion of cell proliferation and inhibition of differentiation during the logarithmic phase of cell growth.
MTA extract promoted the proliferation of hDPCs in part by activating Notch signalling through inhibition of autophagic flux during the early stage and, thus, might potentially induce rapid restoration of injured pulps.
确定自噬和 Notch 信号通路在矿化三氧化物凝聚体（MTA）介导的人牙髓细胞（hDPC）增殖和分化中的作用。
使用胶原酶 I/分散酶 II 消化从人健康磨牙中分离 hDPCs，然后在含 15%胎牛血清的α-MEM 培养基中培养。将 hDPCs 接种于 96 孔板中，通过细胞计数试剂盒检测其活力。实时定量聚合酶链反应（qPCR）用于评估碱性磷酸酶（ALP）、Runx2、Notch1、Hes1 和 Hey1 mRNA 水平。通过 Western blot 定量 Notch1、hes1、LC3 和 p62 蛋白水平。通过免疫荧光测量 LC3 和 Notch1 的共定位。采用双尾学生 t 检验进行统计学分析。
MTA 提取物显著抑制（P < 0.05）自噬流，导致 Notch 降解停滞。这导致对数生长期细胞生长中促进细胞增殖和抑制分化。
MTA 提取物通过在早期阶段通过抑制自噬流激活 Notch 信号通路促进 hDPCs 的增殖，从而可能潜在地诱导受损牙髓的快速修复。
