Center for Exocrine Disorders, Department of Molecular and Cell Biology, Boston University, Henry M. Goldman School of Dental Medicine, Boston, MA, 02118, USA.
Department of Surgery, University of California Los Angeles, Los Angeles, CA, 90095, USA.
Pancreatology. 2020 Mar;20(2):288-292. doi: 10.1016/j.pan.2019.12.020. Epub 2019 Dec 26.
Intrapancreatic activation of digestive proteases, trypsin and chymotrypsin in particular, is a hallmark of pancreatitis. In experimental rodent models, protease activation is routinely measured from pancreatic homogenates using fluorogenic peptide substrates. Here we investigated the optimal conditions for the determination of intrapancreatic trypsin and chymotrypsin activation elicited by a single intraperitoneal injection of cerulein in C57BL/6N mice. We found that these protease assays were significantly improved by using lower amounts of pancreatic homogenate and exclusion of bovine serum albumin from the assay buffer. Furthermore, pancreatic homogenates had to be freshly prepared and assayed; as freezing and thawing stimulated protease activation. Finally, replacement of the widely used Boc-Gln-Ala-Arg-AMC trypsin substrate with Z-Gly-Pro-Arg-AMC reduced the background activity in saline-treated control mice and thereby increased the extent of cerulein-induced trypsin activation. Using the optimized protocol, we reproducibly measured 20-fold and 200-fold increases in the intrapancreatic trypsin and chymotrypsin activity, respectively, in mice given cerulein.
胰腺内消化蛋白酶（尤其是胰蛋白酶和糜蛋白酶）的激活是胰腺炎的一个标志。在实验性啮齿动物模型中，通常使用荧光肽底物从胰腺匀浆中测量蛋白酶的激活。在这里，我们研究了在 C57BL/6N 小鼠单次腹腔注射鹅脱氧胆酸后，测定胰腺内胰蛋白酶和糜蛋白酶激活的最佳条件。我们发现，通过使用较少量的胰腺匀浆并从测定缓冲液中排除牛血清白蛋白，可以显著改善这些蛋白酶测定。此外，胰腺匀浆必须新鲜制备并进行测定；因为冷冻和解冻会刺激蛋白酶的激活。最后，用广泛使用的 Boc-Gln-Ala-Arg-AMC 胰蛋白酶底物代替 Z-Gly-Pro-Arg-AMC，减少了生理盐水处理对照小鼠中的背景活性，从而增加了鹅脱氧胆酸诱导的胰蛋白酶激活程度。使用优化的方案，我们在给予鹅脱氧胆酸的小鼠中分别可重复性地测量到胰腺内胰蛋白酶和糜蛋白酶活性分别增加了 20 倍和 200 倍。
