Abstract:

Objective · To establish a replicative senescence model of human gingival fibroblasts (hGFs), investigate changes in aging related biological characteristics, and provide an efficient cell model for further study on the aging in periodontal diseases. Methods · hGFs were isolated from healthy gingival tissues and cultured with tissue block method in vitro. The tissue source was verified with immunofluorescence. hGFs were continuously cultured and cumulative population doublings (CPD) were calculated and used to draw the curves. Changes in the proliferative capacity of hGFs with CPDs of 10.82, 20.65, 29.52, 42.22, 60.79, and 70.03 were examined with CCK-8. Real-time PCR was used to evaluate changes in the mRNA expression of senescence-related genes p16INK4a and p21Cip1. Results · CPD curves showed that after continuous culture, the CPD value increased gradually and became stable after achieving 70.03. hGFs became flatter and more cell processes appeared with the increase of CPD value. The cell proliferative capacity declined and mRNA levels of p16INK4a and p21Cip1 significantly increased (P=0.000). Conclusion · A replicative senescence model of hGFs is established through continuous culture. CPD curves can reflect the aging of hGFs.

Key words: gingiva, cell aging, tissue block culture techniques, cell proliferation