Stieler Stewart Amy, Freund John M, Blikslager Anthony T, Gonzalez Liara M
Department of Clinical Sciences, North Carolina State University.
Department of Clinical Sciences, North Carolina State University;
J Vis Exp. 2018 May 18(135):57647. doi: 10.3791/57647.
Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ischemia, when the blood supply and therefore oxygen is decreased to the intestine. This leads to intestinal barrier loss and damage to the underlying tissue. Intestinal stem cells reside at the base of the crypts of Lieberkühn and are responsible for intestinal renewal during homeostasis and following injury. Ex vivo cell culture techniques have allowed for the successful study of epithelial stem cell interactions by establishing culture conditions that support the growth of three-dimensional epithelial organ-like systems (termed "enteroids" and "colonoids" from the small and large intestine, respectively). These enteroids are composed of crypt and villus-like domains and mature to contain all of the cell types found within the epithelium. Historically, murine models have been utilized to study intestinal injury. However, a porcine model offers several advantages including similarity of size as well as gastrointestinal anatomy and physiology to that of humans. By utilizing a porcine model, we establish a protocol in which segmental loops of intestinal ischemia can be created within a single animal, enabling the study of differing time points of ischemic injury and repair in vivo. Additionally, we describe a method to isolate and culture the intestinal stem cells from the ischemic loops of intestine, allowing for the continued study of epithelial repair, modulated by stem cells, ex vivo.
肠缺血仍然是人类和兽医患者发病和死亡的主要原因。许多疾病过程都会导致肠缺血，即肠道的血液供应以及由此而来的氧气供应减少。这会导致肠屏障丧失和底层组织受损。肠干细胞位于利伯kühn隐窝底部，在稳态期间和损伤后负责肠道更新。体外细胞培养技术通过建立支持三维上皮器官样系统（分别从小肠和大肠衍生的“肠类器官”和“结肠类器官”）生长的培养条件，成功地研究了上皮干细胞的相互作用。这些肠类器官由隐窝和绒毛样结构域组成，并成熟为包含上皮内所有细胞类型。历史上，小鼠模型一直被用于研究肠道损伤。然而，猪模型具有几个优点，包括与人类在大小以及胃肠道解剖学和生理学方面的相似性。通过使用猪模型，我们建立了一种方案，在单个动物体内创建肠缺血的节段环，从而能够在体内研究缺血性损伤和修复的不同时间点。此外，我们描述了一种从缺血肠环中分离和培养肠干细胞的方法，以便在体外继续研究由干细胞调节的上皮修复。
