| ObjectiveNel-like molecule-1 (Nell-1), with EGF (epidermal cell growth factor)-like coding structure, is a recently discovered as a secreted protein that can specifically induce osteoblast differentiation, bone formation and bone regeneration. Our previous investigations on the expression and localization of Nell-1 during murine molar development indicated that Nell-1 might play a role in odontoblastic differentiation. The aim of the present study was to investigate the effects of Nell-1 on odontoblastic differentiation of human dental pulp cells.Methods1. Cell CultureHuman primary dental pulp cells were isolated and cultured using modified enzyme digestion method.2. Alkaline phosphatase Assay0,50ng/ml, 100ng/ml,150ng/ml Nell-1 protein was added into the cell osteogenic culture media. Cells were cultured for 3 days. And then cells treated with the minimum effective concentration of Nell-1 continued to be cultured for 5,7 days. The total protein was collected and alkaline phosphatase activity was detected with alkaline phosphatase detection kit.3. Real-time PCR and Western blot Human dental pulp cells were cultured with minimum effective concentration Nell-1 for 0,6h,12h and 24h. To detect the changes of RNA levels of odontoblastic differentiation-related molecules such as OPN, DMP-1, OCN and Runx2, quantitative Real-time PCR was performed. The protein level of OPN and DMP-1 was evaluated by Western blot.Results1. Human dental pulp cells were cultured with the modified enzyme method successfully. Passage 3 cells grew fast and were in good shape. Passage 3 cells were used in the follow-up experiments.2. There was no difference in the ALP activity between 0 and 50ng/ml group in 3 days. However, ALP activity was much higher in 100ng/ml treatment than that in 0, 50ng/ml treatment (p<0.05). Notably, ALP activity presented a dose-dependent up-regulation from 100ng/ml to 150ng/ml Nell-1 treated group. So we chose 100ng/ml as the minimum effective concentration for the further study. ALP activity of the Nell-1 treated human dental pulp cells was 1.60 times the amount of the control group in 5 days. In 7 days, the difference was significant between the control and Nell-1 groups (p<0.05).3. The level of OPN, an early marker in the odonblastic stage, in the Nell-1 treated group increased 1.7 times than that of the control group in 6 hours. Such result was extended to stimulation of 12 hours and 24 hours. Similarly, the enhanced expression of DMP-1 by Nell-1 was also obvious. However, OCN and Runx2 had no significant change between the two groups in the testing time. At the protein level, there was no significant difference in the expression of OPN and DMP-1 between control and treated group at 6 hours. Notably, the expression of OPN and DMP-1 at 12 hours in the two groups had obvious difference and the levels of OPN and DMP-1 in the Nell-1 treated group were higher than the control group. When the human dental pulp cells were stimulated by Nell-1 for 24 hours, the expressions of OPN and DMP-1 were enhanced significantly.Conclusions1. Cell morphology was changeful in primary passage. Continuous to passage 3, the cells were spindle and grew well. Human dental pulp cells cultured in vitro kept in a good state and could be used for related experiments.2. As the mineralization time extended, ALP activity rised. ALP activity presented a dose-dependent up-regulation.3. The expression of related odontoblastic markers was evaluated by quantitative Real-Time PCR and Western blot. Nell-1 could accelerate odonblastic differentiation of human dental pulp cells. |
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