Establishment and characterization of primary and subsequent subcultures of normal mouse urothelial cells.
Establishment and characterization of primary and subsequent subcultures of normal mouse urothelial cells.
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DOI：
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发表时间：
2005
期刊：
影响因子：
0.6
通讯作者：
M. E. Kreft;S. Hudoklin;M. Sterle
中科院分区：
文献类型：
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作者：
M. E. Kreft;S. Hudoklin;M. Sterle
在本研究中，我们报道了一种可靠的NMU采收、培养和扩大培养的技术。NMU的获取方法有两种，一种是直接从体内的尿路上皮细胞中获取，另一种是间接从膀胱组织块培养的尿路上皮细胞中获取。原代培养和继代培养在MCDB153+Advanced-DMEM和条件培养基的混合液中繁殖。原代细胞存活需要1×10(5)个活细胞/cm2的初始培养密度，而传代细胞需要较低的培养密度(1×10(4)个活细胞/cm2)。培养的细胞通过上皮样形态和紧密连接蛋白occludin、ZO-1、黏附蛋白E-钙粘蛋白和细胞骨架蛋白7的免疫荧光阳性标记鉴定为尿路上皮细胞。高分化的尿路上皮细胞的标志细胞角蛋白20和尿板蛋白不表达。此外，当尿路上皮细胞以较高的速度复制时，在以后的传代中没有检测到occludin和cell keratin7的免疫荧光标记。尽管使用了来自V79成纤维细胞培养上清液的条件培养液，但在原代培养和继代培养中，NMU表现为基础/中间细胞表型。综上所述，我们证明了NMU的同源长期培养是可以发展的。由于存在强大的转基因工具来操纵小鼠的基因组，我们的发现应该有助于设计小鼠的体外系统，以研究健康和疾病中尿路上皮细胞增殖、分层和分化的控制机制。
In this study, we report a reliable technique for the harvest, cultivation and expansion of monoculture of NMU. The NMU were harvested by two methods, directly from the urothelium in vivo and indirectly from the urothelial outgrowths of bladder explant cultures. Primary cultures and subsequent subcultures were propagated in the mixture of media MCDB 153 and Advanced-DMEM, and conditioned medium. Primary urothelial cells required an initial plating density of 1 x 10(5) viable cells/cm2 for survival, while passaged cells needed lower plating densities (1 x 10(4) viable cells per cm2). The cultured cells were identified as urothelial by their epithelioid morphology and by the positive immunofluorescence labelling of tight junctional proteins, occludin and ZO-1, adherens protein E-cadherin and cytoskeletal protein cytokeratin 7. Markers of highly differentiated urothelial cells, cytokeratin 20 and uroplakins, were not expressed. Furthermore, the immunofluorescence labelling of occludin and cytokeratin 7 was not detected in later passages when urothelial cells replicated at a high rate. In spite of the use of conditioned medium derived from V79 fibroblast cell culture supernatant, the NMU in the primary cultures and subsequent subcultures expressed a basal/intermediate cell phenotype. In conclusion, we demonstrate that homogeneous long-term culture of NMU can be developed. Since powerful transgenic tools exist to manipulate the mouse genome, our findings should help design the mouse in vitro systems for studying the control mechanisms of urothelial cell proliferation, stratification and differentiation in health and disease.