Effect of carboxymethylated chitosan on proliferation, cell cycle and secretion of extracellular matrix in rat nucleus pulposus cells

Abstract

Objective To observe the effect of carboxymethylated chitosan (CMCS) on cell cycle progression,cell cycle protein expression and secretion of extracellular matrix in cultured rat nucleus pulposus cells.Methods Rat nucleus pulposus cells were extracted by using enzymatic digestion and cultured in vitro,and identified by type Ⅱ collagen immunohistochemistry.Nucleus pulposus cells were divided into four groups:control group,and CMCS-treated groups (100,200,500 mg/L for 24 h).Flow cytometry was used to determine the cell cycle progression.The expression of proliferating cell nuclear antigen (PCNA) and Cyclin D was detected by using fluorescence quantitative polymerase chain reaction (FQ-PCR) and Western blotting.The secretion of extracellular matrix (type-2 collagen) was detected by using FQ-PCR.Results The cells in this study were collagen-2 positive by immunohistochemical staining.The expression of PCNA was increased by 12.3％,18.2％,54.2％ and 25.1％,52.7％,66.9％ respectively after treated with 100,200,500 mg/L CMCS as compared with control group (P ＜0.05).The ratio of cells in S phase was increased 1.58,2.34 and 2.61 folder after treated with 100,200,500 mg/L CMCS as compared with control group (P ＜ 0.05).Cyclin D expression was,respectively,increased 1.26,2.15,2.64 and 1.53,1.69,1.83 folder as compared with control group (P ＜ 0.05).FQ-PCR revealed that 100,200,and 500 mg/L CMCS promoted secretion of type Ⅱ collagen in nucleus pulposus cells to 1.67,2.24,2.79 and 1.48,1.65,1.77 folder as compared with control group (P ＜ 0.05).Conclusion CMCS could promote proliferation and PCNA expression,cell cycle progression and Cyclin D expression and secretion of extracellular matrix. Key words: Carboxymethylated chitosan; Nucleus pulposus cells; Proliferation; Cell cycle; Extracellular matrix