Effect of modified chitin on proliferation of human epidermal cells in vitro

Abstract

Objective To investigate the effect of modified chitin on the proliferation of human epidermal cells by in vitro cell culture technique. Methods Human epidermal cells (HaCaT) were added to culture medium containing 0, 10, 100, 500, 1 000, 2 000 and 5 000 mg/L modified chitin, and were cultured for 1, 2, 4, 8 and 14 d, respectively. The treated cells were observed and determined. The effect of modified chitin on the proliferation of HaCaT was evaluated by MTT assay. The content of type Ⅰ and type Ⅲ collagen in cell culture supernatant was examined by ELISA. The protein expression of type Ⅰ collagen COⅡ A1 and proliferating cell nuclear antigen (PCNA) were determined by RT-PCR and Western blotting. Results At the same time, the modified chitin at concentrations of 10 and 100 mg/L showed no significant effect on the proliferation of HaCaT, whereas the modified chitin at the concentrations of 500, 1 000, 2 000 and 5 000 mg/L showed significant promoting effect on cell proliferation (all P<0.05) . With the increase of concentration, the relative increase rate became greater, suggesting that the modified chitin concentration was positively correlated with the promoted proliferation of HaCaT (r=0.855, P=0.03) . At 2 d after culture in 500, 1 000, 2 000 and 5 000 mg/L modified chitin culture solution, the relative proliferation rate of cells began to increase (all P<0.05) . The relative proliferation rate of cells gradually increased over time (all P<0.05) . At the same time, the content of type Ⅰ and type Ⅲ collagen secreted by HaCaT cells began to increase in 500 mg/L modified chitin culture medium (both P<0.05) , and stabilized in 1 000 mg/L modified chitin culture medium. The content of type Ⅰ and type Ⅲ collagen secreted by HaCaT cells began to increase at 2 d after culture in 500, 1 000, 2 000 and 5 000 mg/L modified chitin culture medium (all P<0.05) , and the content of type Ⅰ and type Ⅲ collagen increased gradually over time (both P<0.05) . 500 mg/L modified chitin could significantly increase the mRNA expression levels of type Ⅰ collagen CO Ⅱ A1 and PCNA in HaCaT cells (both P<0.05) , and promote the synthesis and secretion of type Ⅰ collagen COⅡA1 and PCNA protein (both P<0.05) . Conclusion Modified chitin may significantly promote the proliferation of epidermal cells and increase the protein expression levels of type Ⅰ collagen and PCNA, which is expected to play an important role in wound healing. Key words: Modified chitin; Epidermal cells; Cell proliferation; Collagen type Ⅰ; Collagen type Ⅲ; Proliferating cell nuclear antigen