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首页> 外文期刊>American Journal of Physiology >Immunomagnetic enrichment of interstitial cells of Cajal.
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Immunomagnetic enrichment of interstitial cells of Cajal.

机译：卡哈尔间质细胞的免疫磁富集。

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摘要

Disruptions of networks of interstitial cells of Cajal (ICC), gastrointestinal pacemakers and mediators of neurotransmission, can lead to disordered phasic contractions and peristalsis by reducing and uncoupling electrical slow waves. However, detailed analysis of the ICC network behavior has been hampered by their scarcity, limited accessibility in intact tissues, and contamination with other cell types in culture. Our goal was to develop a simple technique to purify ICC from murine gastrointestinal muscles for functional studies. We identified ICC in live small intestinal muscles or primary cell cultures by Kit immunoreactivity using fluorescent antibodies. Because this technique also labels resident macrophages nonspecifically, parallel studies were performed in which nonfluorescent Kit antibodies and macrophages labeled with fluorescent dextran were used for subtractive analysis of ICC. In both groups, Kit-positive cells were tagged with superparamagnetic antibodies and sorted on magnetic columns. Efficacy was assessed by flow cytometry. ICC enrichment from primary cultures and freshly dissociated tissues was approximately 63-fold and approximately 8-fold, respectively. Unlike the cells derived directly from tissues, cells sorted from cultures frequently yielded extensive, nearly homogenous ICC networks on reseeding. Monitoring oscillations in mitochondrial Ca(2+) or membrane potential by imaging revealed spontaneous rhythmicity in these networks. Cells that did not bind to the columns yielded cultures that were depleted of ICC and dominated by smooth muscle cells. In conclusion, immunomagnetic sorting of primary cultures of ICC results in relatively homogenous, functional ICC networks. This technique is less suitable for obtaining ICC from freshly dispersed cells.
机译：Cajal（ICC），胃肠起搏器和神经传递介质的间质细胞网络的破坏，可通过减少和解耦电慢波而导致无序的相收缩和蠕动。但是，对ICC网络行为的详细分析因其稀缺性，在完整组织中的可及性有限以及在培养中受到其他细胞类型的污染而受到阻碍。我们的目标是开发一种从鼠胃肠道肌肉中纯化ICC的简单技术，以进行功能研究。通过使用荧光抗体的试剂盒免疫反应性，我们在活的小肠肌肉或原代细胞培养物中鉴定了ICC。由于该技术还可以非特异性地标记常驻巨噬细胞，因此进行了平行研究，其中将非荧光试剂盒抗体和标记有荧光葡聚糖的巨噬细胞用于ICC的扣除分析。在两组中，用超顺磁性抗体标记Kit阳性细胞，并在磁柱上分类。通过流式细胞术评估功效。来自原代培养物和新鲜离解的组织的ICC富集分别约为63倍和8倍。与直接来自组织的细胞不同，从培养物中分选的细胞经常在重新播种时产生广泛的，几乎同质的ICC网络。通过成像监测线粒体Ca（2+）或膜电位的振荡，揭示了这些网络中的自律性。不与柱结合的细胞产生的培养物被消耗掉ICC，并以平滑肌细胞为主。总之，对ICC原代培养物进行免疫磁分选会产生相对均一的功能性ICC网络。此技术不太适合从新分散的细胞中获得ICC。

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