抑制Notch信号通路对人牙髓细胞的生物学效应

Biological effect of inhibition of Notch signaling pathway on human dental pulp cells

目的 评价抑制Notch信号通路后人牙髓细胞在细胞、代谢酶、基因3个水平的老化指标变化,探讨Notch信号通路对人牙髓细胞老化的作用.方法 采用γ-分泌酶抑制剂3,5-二氟苯乙酰-L-丙氨酰-S苯基甘氨酸t-丁酯抑制Notch信号通路后（抑制组）观察细胞形态,以正常培养基中添加抑制剂的溶剂二甲基亚砜为阴性对照组,检测两组碱性磷酸酶（alkaline phosphatase,ALP）活性、细胞老化相关β-半乳糖苷酶以及老化相关基因p53的表达.结果 抑制Notch信号通路后人牙髓细胞从第10代开始出现细胞形态老化的表现.抑制组自第8代即出现ALP阳性细胞减少,ALP活性下降为（35.36±2.55） U/g,显著低于阴性对照组[（49.76±4.30） U/g]（t=4.989,P=0.008）.抑制组第8代细胞即开始出现老化相关β-半乳糖苷酶阳性染色,第10代时出现大量阳性染色细胞.第10代时,抑制组细胞p53基因相对表达量（1.7±0.4）显著高于阴性对照组（设为1.0）（t=3.581,P=0.012）.结论 抑制Notch信号通路后,人牙髓细胞在细胞、代谢酶、基因3个水平均出现老化前移表现,提示Notch信号通路可以影响人牙髓细胞的生命周期.

Objective To investigate the effect of Notch signaling pathway on human dental pulp cells.Methods The γ-secretase inhibitor N-[N-（3,5-difluorophenacetyl-L-alanyl）]-S-phenylglycine t-butyl ester（DAPT） was applied to inhibit the Notch signaling pathway of human dental pulp cells.The solvent dimethly sulfoxide （DMSO） served as the negative control.Senescence conditions were evaluated by cells morphology changes,the alkaline phosphatase （ALP） expression and its activity,senescenceassociated β-galactosidase（SA-β-Gal） expression and the senescence related gene p53 expression.Results After inhibition of the Notch signaling pathway,morphology changes,including flatter cells,larger plasma area,were seen in the 10th passage human dental pulp cells.ALP expression and activity showed a significant decrease at the 8th passage after inhibition （35.36 ± 2.55） U/g,compared with the negative control group[（49.76 ±4.30） U/g] （t =4.989,P =0.008）.SA-β-Gal-positive cells could be seen as early as the 8th passage and more positive cells were evident at the 10th passage.The relative expression level of p53 gene was elevated in the 10th passage cells（1.7 ±0.4） compared with the negative control group（1.0） （t =3.581,P =0.012）.Conclusions Human dental pulp cells became senescent at earlier passages after inhibition of Notch signaling pathway.Notch signaling pathway may affect life cycle of human dental pulp cells.