Effects of modified chitin on human fibroblasts cultured in-vitro

Abstract

Objective To investigate the effects of different concentrations of modified chitin on human fibroblasts cultured in-vitro. Methods Human fibroblast cell line HFF was cultured in medium containing 0, 10, 100, 500, 1 000, 2 000, 5 000 mg/L modified chitin, and was evaluated at 0, 1, 2, 4, 8 and 14 days of the culture. MTT method was used to evaluation the cytotoxicity of modified chitin, enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of types Ⅰ and Ⅲ collagen in the cell culture supernatant, and the reverse transcription (RT)-PCR and Western blot was used to measure the mRNA and protein expressions of type Ⅰ collagen and proliferating cell nuclear antigen (PCNA). Results MTT assay at days 0, 1, 2, 4, 8 and 14 of culture showed that modified chitin at the concentrations of 10, 100 mg/L did not exhibit any inhibitory effects on proliferation of HFF cells compared with the control group (0 mg/L modified chitin) , while modified chitin at the concentrations of 500, 1 000, 2 000 and 5 000 mg/L significantly inhibited the HFF cell proliferation in a time-and concentration-dependent manner (all P< 0.05). As shown by ELISA assay, treatment of HFF cells with 500, 1 000, 2 000 and 5 000 mg/L modified chitin for 2 d resulted in significantly reduced production of types Ⅰ and Ⅲ collagen in each group of cells compared with the control group (all P<0.05). RT-PCR and Western blot analysis showed that mRNA and protein expression levels of type Ⅰ collagen and PCNA gradually decreased over the 14-day treatment of HFF cells with 500 mg/L modified chitin (all P<0.05). Conclusion Modified chitin can inhibit fibroblast proliferation and expression of type Ⅰ collagen and PCNA in the fibroblast, which may be useful in the prevention and treatment of scarring. Key words: Chitin; Fibroblasts; Collagen type Ⅰ; Proliferating cell nuclear antigen