DAPT对人牙髓细胞Notch信号通路和细胞增殖的影响

Effects of DAPT on proliferation of human dental pulp cells and Notch signaling pathway

目的:探讨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{N-[N-(3,5-difluorophena-cetyl-L-alanyl)]-S-phenylglycine t-butyl ester,DAPT}对人牙髓细胞Notch信号通路是否有抑制作用,及对牙髓细胞增殖能力的影响。方法:收集临床上完整拔除的健康前磨牙或第三磨牙,采用酶消化法进行人牙髓细胞的原代培养和传代培养。从第4代开始在正常培养基中加入终浓度为5μmol/L的γ-分泌酶抑制剂DAPT,选择第4、8、10代细胞为检测细胞,采用实时荧光定量RT-PCR方法和基因芯片检测Notch信号通路相关基因的表达变化,用四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)法测定细胞活性,绘制牙髓细胞增殖曲线,评价牙髓细胞的增殖状况。结果:加入DAPT后,实验组牙髓细胞Notch信号通路的下游基因Hes1、Hey1、NR4A2表达下降,同时,第8、10代牙髓细胞的增殖减慢,与对照组相比,差异有统计学意义。结论:γ-分泌酶抑制剂DAPT能有效抑制体外培养人牙髓细胞的Notch信号通路,使人牙髓细胞增殖减慢,影响细胞的正常生命周期。

Objective：To explore whether the γ-secretase inhibitor N-[ N-（ 3,5-difluorophenacetyl-L-alanyl） ]-S-phenylglycine t-butyl ester （DAPT） could inhibit Notch signaling pathway in human dental pulp ceils, and its effects on the proliferation ability of the cells. Methods： Human dental pulp cells were primarily cultured from healthy premolars or wisdom teeth extracted intactly. The γ-secretase inhibi-tor DAPT （5 μmol/L） was added to the culture medium from passage 4 to the end. The cells of passages 4, 8 and 10 were used as check points in this study. The real-time RT-PCR and RT2 Profiler PCR Array were applied to analyze the expression changes of Notch signaling pathway downstream genes. And the methyl thiazolyl tetrazolium （MTT） method was used to test the proliferation ability of the cells. Results： After DAPT was added, Hesl gene expression level decreased significantly in the cells of passages 4, 8 and 10 as compared with that of the same passage cells in the control group. The relative gene expression ratio （experimental/control） decreased to 0.20 in the cells of passage 10, and the difference was signifi-cant （t = 33. 143, P = 0. 001 ）. The PCR Array results of passage 10 dental pulp cells also showed a decline of Notch signaling pathway downstream genes Hey1 and NR4A2 in the experimental group as com-pared with the control group. The proliferation of passages 8, and 10 experimental cells were slowed down, and the difference was significant. Conclusion： Notch signaling pathway of human dental pulp cells could be inhibited by DAPT effectively. The proliferation of the cells was slowed down by the effect of DAPT, and the normal life cycle of the cells was affected.