Establishment of mouse intestinal myofibroblast cell lines
Establishment of mouse intestinal myofibroblast cell lines
复制标题
DOI：
10.3748/wjg.v19.i17.2629
复制
发表时间：
2013-05-07
影响因子：
4.3
通讯作者：
Sato, Koichi
中科院分区：
文献类型：
--
作者：
Kawasaki, Hideyoshi;Ohama, Takashi;Sato, Koichi
目的：建立小鼠结肠黏膜成肌细胞系，并对其生物学特性进行研究。方法：从去除黏膜上皮细胞和平滑肌层的小鼠结肠黏膜组织中分离出原代培养的成肌细胞。为了永生化，将猴病毒40大T抗原(LmcMF)导入原代IMF。我们还分离了一些未经转染法自发永生化的原代IMF(命名为SmcMF)。为了检查这些细胞的永生化和正常性，我们检测了它们的增殖能力和接触抑制。此外，表征IMF的蛋白表达水平[包括α-平滑肌肌动蛋白(α-SMA)、波形蛋白、结蛋白和类型。免疫相关蛋白[如Toll样受体4(TLR-4)、CD14、MD2、I-kappa Bα和p-p38]，用免疫印迹法检测。结果：细胞生长实验结果显示，LmcMF和SmcMF细胞至少在第20代时均呈对数增殖。此外，接触抑制实验显示LmcMF和SmcMF细胞在达到融合后停止生长。这些数据表明，这两种类型的细胞是永生化的，没有失去生长抑制的接触。此外，Lmcmf和Smcmf与原发IMF一样，均呈纺锤形。Western blotting和免疫荧光分析也检测了关键的肌成纤维细胞蛋白标记物，包括α-SMA、波形蛋白和结蛋白的表达水平。结果表明，这些细胞表达α-SMA和Vimentin，但不表达结蛋白，LmcMF和SmcMF均有表达。胶原蛋白水平低于原发IMF。最后，我们研究了内毒素受体相关蛋白的表达水平，以及细胞对内毒素处理的反应。我们发现TLR4、CD14和MD-2蛋白在LmcMF和SmcMF中以及在原代IMF中都存在，并且这些细胞都对LPS有反应。结论：我们从小鼠结肠粘膜中建立了2个新的IMF细胞系，即LmcMF和SmcMF，这两个细胞都能对内毒素产生反应。(C)2013年白石登。版权所有。
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 alpha-smooth muscle actin (alpha-SMA), vimentin, desmin, and type. collagen] and proteins associated with the immune response [such as toll-like receptor 4 (TLR-4), CD14, MD2, I kappa B alpha, 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 alpha-SMA, vimentin, and desmin, were also examined by the Western blotting and immunofluorescence analyses. Our results show that these cells were positive for alpha-SMA and vimentin, but not desmin, as well as that both LmcMF and SmcMF expressed type. 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. (C) 2013 Baishideng. All rights reserved.