Garcia-Castillo Maria Daniela, Lencer Wayne I, Chinnapen Daniel J-F
Division of Gastroenterology, Boston Children's Hospital, Boston, Massachusetts 02115, USA.
Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Bio Protoc. 2018 Oct 20;8(20). doi: 10.21769/BioProtoc.3049.
Absorption and secretion of peptide and protein cargoes across single-cell thick mucosal and endothelial barriers occurs by active endocytic and vesicular trafficking that connects one side of the epithelial or endothelial cell (the lumen) with the other (the serosa or blood). Assays that assess this pathway must robustly control for non-specific and passive solute flux through weak or damaged intercellular junctions that seal the epithelial or endothelial cells together. Here we describe an cell culture Transwell assay for transcytosis of therapeutic peptides linked covalently to various species of the glycosphingolipid GM1. We recently used this assay to develop technology that harnesses endogenous mechanism of lipid sorting across epithelial cell barriers to enable oral delivery of peptide and protein therapeutics.
肽和蛋白质货物跨单细胞厚度的黏膜和内皮屏障的吸收与分泌，是通过主动内吞和囊泡运输发生的，这种运输将上皮细胞或内皮细胞的一侧（管腔）与另一侧（浆膜或血液）连接起来。评估该途径的检测方法必须有力地控制通过将上皮细胞或内皮细胞密封在一起的薄弱或受损细胞间连接的非特异性和被动溶质通量。在这里，我们描述了一种用于共价连接到各种糖鞘脂GM1的治疗性肽转胞吞作用的细胞培养Transwell检测方法。我们最近使用该检测方法开发了一种技术，该技术利用上皮细胞屏障上脂质分选的内源性机制来实现肽和蛋白质治疗药物的口服递送。
