Loera-Valencia Raúl, Wang Xuan-Yu, Wright George W J, Barajas-López Carlos, Huizinga Jan D
División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica, Camino a la Presa San José 2055, Col. Lomas 4a Sección, C.P.78216, San Luis Potosí, SLP, México,
Cell Mol Biol Lett. 2014 Dec;19(4):601-10. doi: 10.2478/s11658-014-0214-4. Epub 2014 Oct 23.
The interstitial cells of Cajal (ICC) drive the slow wave-associated contractions in the small intestine. A commonly used marker for these cells is c-Kit, but another marker named Ano1 was recently described. This study uses single-cell RT-PCR, qPCR and immunohistochemistry to determine if Ano1 could be reliably used as a molecular marker for ICC in single-cell mRNA analysis. Here, we report on the relationship between the expression of c-Kit and Ano1 in single ICC in culture. We observed that Ano1 is expressed in more than 60% of the collected cells, whereas c-Kit is found only in 22% of the cells (n = 18). When we stained ICC primary cultures for c-KIT and ANO1 protein, we found complete co-localization in all the preparations. We propose that this difference is due to the regulation of c-Kit mRNA in culture. This regulation gives rise to low levels of its transcript, while Ano1 is expressed more prominently in culture on day 4. We also propose that Ano1 is more suitable for single-cell expression analysis as a marker for cell identity than c-Kit at the mRNA level. We hope this evidence will help to validate and increase the success of future studies characterizing single ICC expression patterns.
Cajal间质细胞（ICC）驱动小肠中与慢波相关的收缩。这些细胞常用的标志物是c-Kit，但最近发现了另一种名为Ano1的标志物。本研究使用单细胞逆转录聚合酶链反应（RT-PCR）、定量聚合酶链反应（qPCR）和免疫组织化学来确定在单细胞mRNA分析中Ano1是否可作为ICC可靠的分子标志物。在此，我们报告培养的单个ICC中c-Kit和Ano1表达之间的关系。我们观察到，在收集的细胞中，超过60%表达Ano1，而仅22%的细胞表达c-Kit（n = 18）。当我们对ICC原代培养物进行c-KIT和ANO1蛋白染色时，我们发现在所有制剂中二者完全共定位。我们认为这种差异是由于培养中c-Kit mRNA的调控所致。这种调控导致其转录本水平较低，而Ano1在第4天的培养物中表达更为突出。我们还认为，在mRNA水平上，作为细胞身份的标志物，Ano1比c-Kit更适合用于单细胞表达分析。我们希望这一证据将有助于验证并提高未来表征单个ICC表达模式研究的成功率。
