建立小鼠肠肌成纤维细胞系。
Establishment of mouse intestinal myofibroblast cell lines.
机构信息
Laboratory of Veterinary Pharmacology, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan.
出版信息
World J Gastroenterol. 2013 May 7;19(17):2629-37. doi: 10.3748/wjg.v19.i17.2629.
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.
目的
从鼠结直肠黏膜建立新型肠肌纤维母细胞（IMF）细胞系，并研究其生物学特性。
方法
从去除上皮细胞和平滑肌层的鼠结直肠黏膜组织中分离原代 IMF。为实现永生化，将原代 IMF 用猿猴病毒 40 大 T 抗原（命名为 LmcMF）转染。我们还分离了一些未经转染而自发永生化的原代 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，通过 Western blot 检测了表征 IMF 的蛋白（包括α-平滑肌肌动蛋白（α-SMA）、波形蛋白、结蛋白和 I 型胶原）和与免疫反应相关的蛋白（如 Toll 样受体 4（TLR-4）、CD14、MD2、IκBα和 p-p38）的表达水平。通过免疫荧光染色还检测了几种肌纤维母细胞蛋白标志物的定位。
结果
细胞生长试验结果表明，LmcMF 和 SmcMF 细胞的对数生长期至少可延续至传代 20 代。此外，接触抑制试验表明，LmcMF 和 SmcMF 细胞在达到汇合后停止生长。这些数据表明，这两种类型的细胞在失去生长接触抑制的情况下被永生化。此外，LmcMF 和 SmcMF 与原代 IMF 一样，呈梭形外观。通过 Western blot 和免疫荧光分析还检测了关键肌纤维母细胞蛋白标志物的表达水平。结果表明，这些细胞呈α-SMA 和波形蛋白阳性，但结蛋白阴性，并且 LmcMF 和 SmcMF 的 I 型胶原表达水平均低于原代 IMF。最后，我们研究了脂多糖（LPS）受体相关蛋白的表达水平以及细胞对 LPS 处理的反应。结果发现，TLR4、CD14 和 MD-2 蛋白存在于 LmcMF 和 SmcMF 以及原代 IMF 中，并且所有这些细胞均对 LPS 有反应。
结论
我们从鼠结直肠黏膜建立了 2 种新型 IMF 细胞系，即 LmcMF 和 SmcMF，它们均能对 LPS 作出反应。
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