Effects of bFGF on the Modulation of Apoptosis in Gingival Fibroblasts with Different Host Ages

Abstract

The purpose of this study was to investigate the effects of basic fibroblast growth factor (bFGF) treatment on the proliferation and apoptosis of cultured gingival fibroblasts (GFs). Human GFs were isolated from the palatal gingival tissues of 16 healthy volunteers ranging in the age from 9 to 35 years old. Cultured GFs were subjected to the analyses for cell proliferation by ELISA assay, gene expression by RT-PCR analysis, and apoptosis potency by caspase-3 assay. The cell proliferation activity and gene expression of type-I collagen and caspase-3 activity were enhanced significantly by the treatment with bFGF in cultured GFs. Furthermore, the activity of caspase-3 in cultured GFs from young subjects was significantly higher than that in GFs from adults. It is shown that bFGF significantly enhances the gene expression of type-I collagen in cultured fibroblasts from human gingival tissues. It also demonstrated that bFGF modulates the apoptosis of periodontal fibroblasts, and the effect is higher in young subjects, indicating a significant role of bFGF in the prevention of scar formation during wound healing.

Figure 1

Proliferation of cultured human GFs induced by addition of bFGF. GFs were seeded at a density of 1.0 × 10³ cells/well. Cell proliferation of cultured GFs with 0 (a), 0.5 (b), and 1.0 (c) ng/mL bFGF was evaluated by measuring BrdU incorporated into the DNA of proliferating cells 3, 5, 7, and 9 days after seeding. Error bars indicate a standard deviation. N = 8,  *P < 0.05,  **P < 0.01.

Figure 2

Changes in the gene expression of fibronectin-1 and type-I collagen during differentiation in cultured GFs. Total RNA was extracted from GFs as the cells became confluent; GFs were incubated with bFGF of 0.5 or 1.0 ng/mL for 24 hrs. The mRNA expression levels of fibronectin-1 (a) and type-I collagen (b) were determined by means of a real-time PCR analysis, normalized relative to the expression of GAPDH, and depicted as the rate of change in gene expression. Error bars indicate a standard deviation. N = 8,  *P < 0.05,  **P < 0.01.

Figure 3

Effect of the activation of bFGF on the induction of apoptosis in cultured human GF. (a) Comparison of caspase-3 activity between 70% confluent and 100% confluent groups. (b) Time course of caspase-3 activity in 100% confluent group. Error bars indicate a standard deviation. N = 8,  *P < 0.05, **P < 0.01.

Figure 4

Effect of bFGF on apoptosis in rat palate. At 10 days after the excision, the head was resected under deep anesthesia with diethyl ether and fixed by immersion. Paraffin sections of the rat palate 6 ((a), (b)) and 12 weeks ((c), (d)) were stained with ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit and observed by light microscopy. (a), (c): control group; (b), (d): bFGF injection group. The scale bars represent 25 μm.