激活Notch信号通路可延迟人牙髓细胞的老化表现

摘要/Abstract

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。

关键词: 受体, Notch, 信号传导, 细胞衰老, 牙髓

Abstract: Objective：To establish model of dental pulp cells with activated Notch signaling pathway, and investigate the effect of activating Notch signaling pathway on senescence of human dental pulp cells in vitro. Methods: Human dental pulp cells were isolated, cultured as usual, and used from the 4^th passage. The cells were divided into the activated group and the negative control group. In the activated group, the way of coating dishes with Jagged1 protein (10 mg/L) was used to activate Notch signaling pathway. The negative control group cells received no treatment. In the 4^th, 8^th, and 10^th passages, the expression levels of the Notch signaling pathway downstream gene Hes1 were verified by real-time quantitative PCR (RT-qPCR). The cell changes after activating Notch signaling pathway were observed at three levels: (1) The cell morphology changes were observed through invert phase contrast microscope. The cell activity was detected with MTT assay. (2) The alkaline phosphatase (ALP) expression and its activity, and senescence-associated β-galatosidase (SA-β-Gal) expression were observed with the kit. (3) The expression changes of senescence related genes were verified using RT-qPCR. The difference between the negative control group and the activated group was analyzed using student’s t test. Results: The expression level of the downstream gene Hes1 of Notch signaling pathway increased after coating the dishes with Jagged1 protein, indicating the establishment of the model of dental pulp cells with activated Notch signaling pathway. Compared with the negative control group, the aging cells of the activated group appeared relatively late. In the 8th and 10th passage, the cell activity increased. In the 10th passage, ALP activity increased, but SA-β-Gal expression decreased. p16 gene expression decreased in each passage, and p53 gene expression decreased in the 8th and 10th passages. Conclusion: Jagged1 could activate Notch signaling pathway effectively. Through activating Notch signaling pathway, the dental pulp cells showed a trend of senescence delay at different levels, such as cell morphology, metabolic enzyme expressions and related gene expressions.

Key words: Receptors, Notch, Signal transduction, Cell aging, Dental pulp

常思佳，邹晓英，庄姮，岳林，高学军. 激活Notch信号通路可延迟人牙髓细胞的老化表现[J]. 北京大学学报（医学版）, 2014, 46(1): 5-11.

CHANG Si-jia, ZOU Xiao-ying, ZHUANG Heng, YUE Lin, GAO Xue-jun. Notch activation delayed ageing of human dental pulp cells[J]. Journal of Peking University(Health Sciences), 2014, 46(1): 5-11.

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