激活Notch信号通路可延迟人牙髓细胞的老化表现

发布时间：2018-06-22 08:04

本文选题：受体 + Notch　；参考：《北京大学学报(医学版)》2014年01期

【摘要】：目的:建立Notch信号通路激活的人牙髓细胞模型,探讨激活Notch信号通路对人牙髓细胞老化的影响。方法:体外培养人牙髓细胞,从第4代开始实验,将牙髓细胞分为激活组和阴性对照组。激活组采用10 mg/L的Jagged1蛋白包被培养皿的方法激活Notch信号通路,阴性对照组的培养皿不做处理。以实时定量PCR(real-time quantitative PCR,RT-qPCR)检测第4代、第8代、第10代Notch信号通路下游基因Hes1的表达水平。分三个水平观察Notch信号通路激活后人牙髓细胞的变化:(1)细胞水平,用倒置显微镜观察细胞形态的变化,用MTT法检测细胞活力改变;(2)代谢酶水平,用碱性磷酸酶(alkaline phosphatase,ALP)染色及活性测定试剂盒检测ALP的表达和活性,用衰老相关β-半乳糖苷酶染色方法检测阳性细胞百分率;(3)基因水平,用RT-qPCR检测老化相关基因p53和p16的表达。t检验比较激活组和阴性对照组各指标的差异。结果:采用Jagged1蛋白包被培养皿后,Notch信号通路下游基因Hes1表达增高,表明建立了Notch信号通路激活的人牙髓细胞模型。激活组牙髓细胞与阴性对照组牙髓细胞相比,较晚出现形态显现衰老的细胞,第8代、第10代细胞活力升高,ALP表达量升高;第10代ALP活性升高,β-半乳糖苷酶表达下降;各代p16基因表达水平均降低,第8代、第10代p53基因表达水平降低。结论:Jagged1蛋白可有效激活Notch信号通路,激活Notch信号通路后,人牙髓细胞在细胞水平、代谢酶水平、基因水平均表现出老化延迟的趋势。
[Abstract]:Aim: to establish a human dental pulp cell model with Notch signaling pathway activation and to investigate the effect of Notch signal pathway activation on human dental pulp cell aging. Methods: human dental pulp cells were cultured in vitro. Dental pulp cells were divided into activated group and negative control group. In the activation group, the Notch signaling pathway was activated by using a 10 mg / L JaggeD1 protein coated with a petri dish, but not in the negative control group. The expression of Hes1 gene downstream of Notch signaling pathway in the 4th, 8th and 10th generation was detected by real-time quantitative PCR RT-qPCR. The changes of human dental pulp cells after Notch signal pathway activation were observed at three levels: (1) cell level, cell morphology by inverted microscope, cell viability by MTT assay, and (2) metabolic enzyme level. The expression and activity of alkaline were detected by alkaline phosphatase staining and activity assay kit, and the percentage of positive cells was detected by senescence associated 尾 -galactosidase staining. (3) Gene level. RT qPCR was used to detect the expression of p53 and p16 genes. T test was used to compare the differences between the activation group and the negative control group. Results: the expression of Hes1 gene downstream of Notch signaling pathway was increased after coated with Jagged1 protein, which indicated that the human dental pulp cell model was established with Notch signaling pathway activated. Compared with the dental pulp cells of the negative control group, the activated dental pulp cells appeared the aging cells later, the activity of ALP increased and the expression of 尾 -galactosidase decreased in the 8th generation, the 10th generation and the 10th generation respectively, and the activity of ALP increased and the expression of 尾 -galactosidase decreased. The expression level of p16 gene decreased in all generations, but decreased in the 8th generation and 10th generation. Conclusion the cell level, metabolic enzyme level and gene level of human dental pulp cells showed a tendency of delayed aging after the activation of Notch signaling pathway by 1: Jagged1 protein.
【作者单位】：北京大学口腔医学院·口腔医院牙体牙髓科;
【基金】：国家自然科学基金(30801289)资助~~
【分类号】：R329.26

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