Glaeser Hartmut, Drescher Siegfried, van der Kuip Heiko, Behrens Christoph, Geick Anke, Burk Oliver, Dent John, Somogyi Andrew, Von Richter Oliver, Griese Ernst-Ulrich, Eichelbaum Michel, Fromm Martin F
Dr Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany.
Clin Pharmacol Ther. 2002 Mar;71(3):131-40. doi: 10.1067/mcp.2002.121370.
Intestinal metabolism and transport are now recognized as protective barriers against orally ingested xenobiotics, including drugs. However, in vitro studies of the expression and function of intestinal proteins are hampered by the limited availability of human intestinal tissues. Because enterocytes are constantly shed in large numbers into the gut lumen, this study investigated whether these cells could be collected with a multilumen perfusion catheter and whether they are functionally active.
In healthy volunteers, a 20-cm isolated jejunal segment was generated with the perfusion catheter by inflating 2 balloons with air. Shed cells were characterized by fluorescence-activated cell sorting analysis for leukocyte-specific CD45 and enterocyte-specific villin, as well as for apoptosis. Homogenates of the cells were used for reverse transcriptase polymerase chain reaction and Western blotting. Cytochrome P450 enzyme activity was determined with the calcium channel blocker verapamil as a substrate.
On average, 4.83 mg protein and 56.23 million cells were collected from a 20-cm segment during 2 hours. A total of 84.2% of the cells were positive for enterocyte-specific villin, and only 1.6% of the collected cells were positive for CD45. The majority of cells (65.3%) were not in early or late apoptosis or necrosis. In all volunteers, drug-metabolizing enzymes (such as members of the cytochrome P450 family) could be detected as both messenger ribonucleic acid and proteins. Consistent with expression data, formation of verapamil metabolites catalyzed by CYP3A4 and CYP2C was shown.
The majority of shed human enterocytes collected with a multilumen perfusion catheter were still functionally active and not apoptotic. Harvesting of spontaneously shed enterocytes provides a new tool for studies on expression and function of intestinal proteins.
肠道代谢和转运现被认为是抵御口服摄入的包括药物在内的外源性物质的保护屏障。然而，人肠道组织获取有限阻碍了肠道蛋白质表达和功能的体外研究。由于肠上皮细胞不断大量脱落至肠腔，本研究调查了能否使用多腔灌注导管收集这些细胞以及它们是否具有功能活性。
在健康志愿者中，通过向两个气囊充气，使用灌注导管构建一段20厘米长的孤立空肠段。通过荧光激活细胞分选分析对脱落细胞进行特征鉴定，检测白细胞特异性CD45、肠上皮细胞特异性绒毛蛋白以及细胞凋亡情况。细胞匀浆用于逆转录聚合酶链反应和蛋白质印迹分析。以钙通道阻滞剂维拉帕米为底物测定细胞色素P450酶活性。
在2小时内，从一段20厘米长的肠段平均收集到4.83毫克蛋白质和5623万个细胞。总共84.2%的细胞肠上皮细胞特异性绒毛蛋白呈阳性，而收集到的细胞中只有1.6%的CD45呈阳性。大多数细胞（65.3%）未处于早期或晚期凋亡或坏死状态。在所有志愿者中，均可检测到药物代谢酶（如细胞色素P450家族成员）的信使核糖核酸和蛋白质。与表达数据一致，显示了由CYP3A4和CYP2C催化的维拉帕米代谢产物的形成。
用多腔灌注导管收集的大多数脱落的人肠上皮细胞仍具有功能活性且未凋亡。收集自发脱落的肠上皮细胞为肠道蛋白质表达和功能研究提供了一种新工具。
