An in vitro system for the study of epithelial bicarbonate secretion

描述（申请人提供）：碳酸氢盐分泌在十二指肠、气道、胰腺、肝脏、男性生殖系统和肾脏的生理功能中起着至关重要的作用。碳酸氢盐分泌的损害，如囊性纤维化所表现的，可在部分或全部这些器官中产生严重的病理。尽管经过几十年的深入研究，碳酸氢盐分泌的机制仍未完全了解。部分问题在于其研究固有的方法，可能涉及肠、胰管和肾小管灌注，以及通常在活体动物或完整组织中使用的间接方法。所有这些方法的缺点是费用过高，操作繁琐且耗时，实验动物的使用需要相当大的费用和近乎禁止的监管要求，以及所研究组织的异质性和缺乏活力。体外分泌碳酸氢盐的系统虽然克服了这些障碍，但尚未开发出来。我们建议基于常见的上皮细胞系开发一种碳酸氢盐分泌系统，该系统具有与碳酸氢盐分泌完整上皮相似的属性，因此可能具有可测量的碳酸氢盐分泌。我们的方法包括在半透性支架上培养Caco-2肠细胞，或者MDCK或其他上皮来源的培养细胞，然后可以将其安装在灌注室中，除了细胞内pH值的比例测量外，还可以测量碳酸氢盐分泌，以及单层的短路电流。以这种方式，我们计划生成数据，使我们能够测试几个关键假设。公共卫生相关性：肠道分泌碳酸氢盐（小苏打），以中和胃分泌的酸。其他重要器官如胰腺、肝脏、肺和男性生殖系统也会分泌碳酸氢盐。在囊性纤维化疾病中发现的碳酸氢盐分泌紊乱会导致严重的问题，如肺病、胰腺炎、肠梗阻和男性不育。碳酸氢盐分泌机制的研究一直受阻于缺乏合适的模型，而不是使用活体动物。我们提出了一个适合研究碳酸氢盐分泌的基于细胞培养的系统，这将有助于我们和其他研究者研究碳酸氢盐是如何分泌的。有了这些知识，我们希望进一步了解为什么碳酸氢盐分泌问题会导致囊性纤维化等严重疾病。

DESCRIPTION (provided by applicant): Bicarbonate secretion plays an essential role in the physiological functioning of the duodenum, airway, pancreas, liver, male reproductive system, and kidney. Impairment of bicarbonate secretion, such as is manifest in the disease cystic fibrosis, can produce severe pathology in some or all of these organs. The mechanism of bicarbonate secretion is still not fully understood, despite decades of intensive study. Part of the problem is the methodology inherent in its study, which can involve intestinal, pancreatic duct, and renal tubule perfusion, and indirect methods, usually in live animals or in intact tissue. All of these methods suffer from excessive expense, cumbersome and time-consuming procedures, the use of laboratory animals which entails considerable expense and near- prohibitive regulatory requirements, and heterogeneity and lack of viability of the tissues studied. A bicarbonate-secreting in vitro system, although overcoming most of these obstacles, has not yet been developed. We propose to develop a bicarbonate secreting system based on commonly studied epithelial cell lines that have attributes similar to bicarbonate secreting intact epithelia and are thus likely to have measurable bicarbonate secretion. Our methodology includes culturing Caco-2 intestinal cells, or alternatively MDCK or other epithelial-derived cultured cells, on semi-permeable supports that can then be mounted in a perfusion chamber that enables the measurement of bicarbonate secretion, in addition to ratiometric measurement of intracellular pH, and short-circuit current across the monolayer. In this fashion, we plan to generate data that will enable us to test several key hypotheses. PUBLIC HEALTH RELEVANCE: Bicarbonate (baking soda) is secreted by the intestine in order to neutralize acid that is secreted by the stomach. Bicarbonate is also secreted by other important organs such as the pancreas, liver, lung, and male reproductive system. Disorders of bicarbonate secretion, as are found in the disease cystic fibrosis, can cause severe problems such as lung disease, pancreatitis, intestinal obstruction, and male infertility. Study of the mechanism of bicarbonate secretion has been hampered by the lack of a suitable model other than the use of live animals. We propose a cell-culture-based system suitable for the study of bicarbonate secretion that will help us and other investigators study how bicarbonate is secreted. With this knowledge, we hope to further understand why problems with bicarbonate secretion can lead to such sever diseases such as cystic fibrosis.