Methods: Epithelial cells from the porcine epithelial rest of Malassez (ERM) were isolated from the periodontal ligament and cultured by the method described by Brunette et al. The cells were cultured in DMEM supplemented with 10% FBS or keratinocyte growth medium (KGM). In order to observe the effect of RKI on ERM cells, 5, 10 and 50 µM of Y-27632 (Sigma) was added to the culture systems. The cells cultured without Y-27632 were used as controls. Telomerase reverse transcriptase (TERT) expression was used as a marker for life-span extension. P16 was used as a marker for senescence. The expression levels of TERT, p16, Amelogenin (Ag), Ameloblastin (Ab) and Enamelysin (En) were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR. Data from the real-time PCR were analyzed using Mann-Whitney’s U-test.
Results: The expression level of TERT was significantly higher in the cells stimulated with RKI than in the control after 35th passages (p<0.05). The expression level of p16 was significantly lower in the cells stimulated with RKI than in the control after 40th passages (p<0.05). The expression levels of Ag and Ab were significantly higher in the cells stimulated with RKI than in the control (p<0.05), whereas the expression level of En was significantly lower in the cells stimulated with RKI than in the control (p<0.05).
Conclusions: The present study suggests that Rho kinase inhibitor inhibit senescence of ERM cells.