Liu H J, Wang T, Li Q M, Guan X Y, Xu Q
Guanghua School of Stomatology & Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, China.
Int Endod J. 2015 Oct;48(10):976-85. doi: 10.1111/iej.12392. Epub 2014 Nov 12.
To investigate the role of p300 in the regulation of proliferation and odontogenic differentiation of human dental pulp cells (HDPCs).
The recombinant lentiviral vector pshRNA-copGFP was used to knock-down p300 expression in HDPCs. Protein level of acetylated H3 was detected. The proliferation of HDPCs was measured using the CCK8 assay. The cell cycle and apoptosis were analysed using flow cytometry and TUNEL staining, respectively. The expression levels of Cdc25A, p21(waf1) and the cleaved products of caspase 3 and caspase 7 were determined utilizing real-time quantitative polymerase chain reaction and Western blotting analysis. The alkaline phosphatase (ALP) activity was measured, and the formation of mineralized nodules was assessed using alizarin red staining after the induction of odontogenic differentiation of HDPCs. The expression levels of the odontogenic differentiation markers DMP-1, DSPP and DSP were detected utilizing real-time quantitative polymerase chain reaction and Western blotting analysis.
After p300 was knocked down in HDPCs, p300 was significantly down-regulated at both the mRNA and protein levels, and histone H3 acetylation was reduced. The proliferation capacity of HDPCs was suppressed in p300 knock-down groups. The cells were arrested in the G0/G1 phase of the cell cycle, and cell apoptosis was triggered. ALP activity, the formation of mineralized nodules and the expression levels of DMP-1, DSPP and DSP were all decreased in p300-knock-down HDPCs undergoing odontogenic differentiation.
Knocking down p300 restrains the proliferation and odontogenic differentiation potentiality of HDPCs.
探讨p300在调控人牙髓细胞（HDPCs）增殖和牙源性分化中的作用。
采用重组慢病毒载体pshRNA-copGFP敲低HDPCs中p300的表达。检测乙酰化H3的蛋白水平。使用CCK8法检测HDPCs的增殖情况。分别采用流式细胞术和TUNEL染色分析细胞周期和细胞凋亡。利用实时定量聚合酶链反应和蛋白质印迹分析测定Cdc25A、p21（waf1）以及半胱天冬酶3和半胱天冬酶7的裂解产物的表达水平。测量碱性磷酸酶（ALP）活性，并在诱导HDPCs进行牙源性分化后，使用茜素红染色评估矿化结节的形成情况。利用实时定量聚合酶链反应和蛋白质印迹分析检测牙源性分化标志物DMP-1、DSPP和DSP的表达水平。
在HDPCs中敲低p300后，p300在mRNA和蛋白水平均显著下调，组蛋白H3乙酰化水平降低。p300敲低组中HDPCs的增殖能力受到抑制。细胞停滞在细胞周期的G0/G1期，并引发细胞凋亡。在进行牙源性分化的p300敲低的HDPCs中，ALP活性、矿化结节的形成以及DMP-1、DSPP和DSP的表达水平均降低。
敲低p300可抑制HDPCs的增殖和牙源性分化潜能。
