Original Article
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. May 7, 2013; 19(17): 2629-2637
Published online May 7, 2013. doi: 10.3748/wjg.v19.i17.2629
Establishment of mouse intestinal myofibroblast cell lines
Hideyoshi Kawasaki, Takashi Ohama, Masatoshi Hori, Koichi Sato
Hideyoshi Kawasaki, Takashi Ohama, Koichi Sato, Laboratory of Veterinary Pharmacology, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan
Masatoshi Hori, Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, the University of Tokyo, Tokyo 113-8657, Japan
Author contributions: Ohama T and Sato K designed the research; Kawasaki H, Hori M and Ohama T performed the experiments; Kawasaki H and Ohama T analyzed the data; and Kawasaki H, Ohama T and Sato K wrote the paper.
Supported by Uehara Memorial Foundation and Mishima Kaiun Memorial Foundation; A Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology
Correspondence to: Koichi Sato, PhD, Laboratory of Veterinary Pharmacology, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan. k-sato@yamaguchi-u.ac.jp
Telephone: +81-83-9335905 Fax: +81-83-9335905
Received: November 13, 2012
Revised: December 3, 2012
Accepted: January 11, 2013
Published online: May 7, 2013
Processing time: 174 Days and 16.9 Hours
Abstract

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.

Keywords: Cell line; Colon; Lipopolysaccharide; Mouse; Myofibroblast