Establishment of mouse intestinal myofibroblast cell lines

摘要

AIM:To establish novel intestinal myofibroblast (IMF) cell lines from mouse colonic mucosa and investigate their biological characters. METHODS:Primary IMFs were isolated from mucosal tissues of mouse colon that was denuded of epithelial cells and smooth muscle layer. For immortalization, primary IMFs were transfected with simian virus 40 large T antigen (designated as LmcMF). We also isolated some primary IMFs that spontaneously became immortalized without transfection (designated as SmcMF). To check immortality and normality of these cells, we examined their proliferative ability and contact inhibition. Moreover, the expression levels of proteins characterizing IMFs [including α-smooth muscle actin (α-SMA), vimentin, desmin, and type Ⅰ collagen] and proteins associated with the immune response [such as toll-like receptor 4 (TLR-4), CD14, MD2, IκBα, and p-p38] were determined by Western blotting. The localization of several myofibroblast protein markers was also detected by immunofluorescence staining. RESULTS:The cell growth assay results show that both LmcMF and SmcMF cells proliferated logarithmically at least up to passage 20. In addition, the contact inhibition assays show that LmcMF and SmcMF stopped growing after the cells reached confluence. These data suggest that these 2 types of cells were immortalized without losing contact inhibition of growth. Moreover, both LmcMF and SmcMF, like primary IMFs, showed spindle-shaped appearance. The expression levels of key myofibroblast protein markers, including α-SMA, vimentin, and desmin, were also examined by the Western blotting and immunofluorescence analyses. Our results show that these cells were positive for α-SMA and vimentin, but not desmin, as well as that both LmcMF and SmcMF expressed type Ⅰ collagen at a lower level than primary IMFs. Finally, we investigated the expression level of lipopolysaccharide (LPS) receptor-related proteins, as well as the response of the cells to LPS treatment. We found that the TLR4, CD14, and MD-2 proteins were present in LmcMF and SmcMF, as well as in primary IMFs, and that all these cells responded to LPS. CONCLUSION:We established 2 novel IMF cell lines from mouse colonic mucosa, namely, LmcMF and SmcMF, both of which were able to respond to LPS.