Objectives: To observe proliferation and senescence of human dental pulp cells after blocking Notch signaling pathway in order to discuss senescence mechanism of the cells.
Methods: Human dental pulp cells were cultured, adding DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycinet-butyl ester, gamma-secretase inhibitor, 5µmol/L) to block Notch signaling pathway. Cells were seeded into 96-well plates at 3*103 cells/well and 6 replicated wells were set for each group. Proliferation of the cells was evaluated by growth curve using MTT assay. The optical density value (OD) of control group and DAPT group cells were examined every 48h and data was statistically analyzed by Student's t test. Senescence condition of the cells was observed by the expression of senescence-associated b-galactosidase (SA-b-Gal) using b-galactosidase stain.
Results: Proliferation of the cells treated by DAPT was decreased compared with the control group, the difference between the two groups was statistically significant (P<0.05). SA-b-Gal stain positive cells were seen in the DAPT group, but no positive stain cells were found in the control group.
Table 1 Effects of blocking Notch signaling pathway on proliferation of human dental pulp cells.(MEAN±SD)
days | Control group(OD) | DAPT group(OD) |
1 | 0.199±0.018 | 0.189±0.015 |
3 | 0.249±0.029 | 0.199±0.020* |
5 | 0.292±0.030 | 0.192±0.015* |
7 | 0.320±0.019 | 0.238±0.039* |
9 | 0.380±0.028 | 0.255±0.035* |
11 | 0.537±0.051 | 0.307±0.040* |
13 | 0.644±0.056 | 0.347±0.041* |
15 | 0.742±0.098 | 0.354±0.066* |
17 | 0.814±0.091 | 0.374±0.039* |
*represents P<0.05.
Conclusion: Blocking Notch signaling pathway by DAPT can decrease proliferation and increase senescence of human dental pulp cells. The results indicate that Notch signaling pathway could be correlated to senescence of human dental pulp cells.