Objective: To evaluate transcriptional activity in vitro of genes involved in cell-cycle (i.e. E2F1, PI3K) and apoptosis (i.e. p53, Bcl-2 and Bax) on human gingival fibroblasts (HGF) stimulated with PRP at different times after its preparation.
Methods: Primary cultures of HGF were obtained from gingival tissue from healthy volunteers whose informed consent was approved by ethics committee of Javeriana University. PRP was prepared using blood samples of healthy adults from a blood bank by a two-step centrifugation procedure, followed by activation with 10% CaCl2 and stored at -20°C until used. The effect of time after PRP (5% and 10%) preparation with and without FBS on apoptosis and cell-cycle genes expression was evaluated by RT-PCR at 0,24,48,72 and 96 hours. Control group was not stimulated with PRP. Statistically significant differences between groups were determined using Kruskal-Wallis test (p≤ 0,0001).
Results: In general apoptotic and cell-cycle genes from HGF exposed to PRP were up-regulated compared with the un-stimulated control group. This response was irrespective of PRP concentration. HGF exposed to PRP 48,72 and 96h after its preparation showed higher expression of apoptotic genes, whereas cell-cycle genes activation was associated with cells incubated with PRP prepared within the first 24h. Over expression of Bcl2, pI3k and E2F (cell survival) and activation of p53 in HGF treated with PRP and FBS was observed. In contrast, cells treated with PRP alone showed down-regulation of genes involved in cell proliferation, and apoptosis genes were up-regulated.
Conclusion: Time after PRP preparation appears to differentially induce expression of cell-cycle and apoptotic genes in HGF, especially in presence of FBS.