연골세포의 탈분화 과정에서 제2형 교원질 발현에 미치는 유전자 메틸화의역활

Abstract

Purpose: We explored the possibility of DNA methylation as a mechanism of loss of type II collagen expression in dedifferentiating chondrocytes by culturing in monolayer. Materials and Methods: Dedifferentiation was induced by low density subeulturing primary porcine chon￢drocytes in vitro. The mRNA expression of Type I collagen, Type II collagen and DNA methyltransferase (DNMT) was measured by RT-PCR. Induction of redifferentiation in dedifferentiated chondrocytes was performed in 3-dimensional alginate bead culture system As stimulating factors for reexpression of genes in dedifferentiated chondrocytes, 10 nglml TGF-f31 and 5,uM 5-azacytidine were used. Results: Type II collagen mRNAs was expressed strongly in freshly isolated cells but had decreased in monolayer cultured cells after 3 weeks up to 40%. In contrast, type I collagen expression was increased from 21 days and kept increasing during the 86 days of study. After treatment of 5 pM 5-azacytidine, fibroblast like morphology was changed to round shape such as traditional chondrocyte morphology at day 4. At day 10, type Ii collagen expression was increased by 5-azacytidine and TGF-(31 marginally and also integrin f31 expression was increased in all groups. RT-PCR analysis demonstrated that DNMT3A expression increased in dedifferentiating chondrocytes when compared with control cells for 40 days. Conclusion: Loss of type II collagen mRNA expression and increase of DNMT 3A expression were showed similar patterns during dedifferentiation. These results suggest that type II collagen gene expression may be influenced by DNA methylation. As stimulating factors, TGF-f31 and 5-azacytidine have potential activity to increase the type II collagen expression in alginate culture system.