Rose Natisha L, Palcic Monica M, Lakey Jonathan R T
Department of Surgery, Surgical-Medical Research Institute, University of Alberta, Edmonton, Alberta, Canada.
Pancreas. 2003 Aug;27(2):167-73. doi: 10.1097/00006676-200308000-00010.
Recent evidence has suggested that inconsistencies in human islet yield and viability after collagenase digestion is attributed to the activation of endogenous enzymes of the cadaveric donor pancreas. A study of the enzyme kinetics of serine proteases throughout human islet isolations showed a significant increase in activity levels throughout the digestion period. Following the digestion, it is important to further inhibit these enzymes by the addition of an inhibitor to the dilution media.
To report the levels of endogenous pancreatic enzymes remaining after human islet isolation and the effects of three potential enzyme inhibitors on the proteases.
Human albumin, fetal calf serum, and the protease inhibitor aprotinin were incubated with the trypsin, chymotrypsin, elastase, and collagenase and were assayed for activity.
Results at the final stage indicated that chymotrypsin retained 21.0 +/- 7.5% (mean +/- SE; n = 20) of the activity observed at the conclusion of the enzymatic digestion phase of the isolation process, whereas trypsin, elastase, and collagenase retained 3.0 +/- 1.5%, 2.1 +/- 0.6%, and 3.9 +/- 0.9%, respectively. Fetal calf serum and aprotinin showed strong inhibitory effects against bovine pancreatic trypsin; however, they showed a weak inhibitory effect against elastase. Supplementation with aprotinin failed to inhibit human chymotrypsin and elastase. Human albumin showed minimal inhibition and was shown to serve only as a competitive inhibitor. No inhibition to collagenase was observed with human albumin, fetal calf serum, or aprotinin.
This study clearly demonstrates that low amounts of endogenous pancreatic enzymes remain active throughout the human islet isolation process and that the added inhibitors at the end of the isolation process are not fully effective at inhibiting the enzymes.
最近的证据表明，胶原酶消化后人胰岛产量和活力的不一致归因于尸体供体胰腺内源性酶的激活。一项关于人胰岛分离过程中丝氨酸蛋白酶的酶动力学研究表明，在整个消化期间活性水平显著增加。消化后，通过向稀释培养基中添加抑制剂来进一步抑制这些酶很重要。
报告人胰岛分离后残留的内源性胰腺酶水平以及三种潜在酶抑制剂对蛋白酶的影响。
将人白蛋白、胎牛血清和蛋白酶抑制剂抑肽酶与胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶和胶原酶一起孵育，并检测其活性。
最终阶段的结果表明，胰凝乳蛋白酶保留了分离过程酶消化阶段结束时观察到的活性的21.0±7.5%（平均值±标准误；n = 20），而胰蛋白酶、弹性蛋白酶和胶原酶分别保留了3.0±1.5%、2.1±0.6%和3.9±0.9%。胎牛血清和抑肽酶对牛胰蛋白酶显示出强烈的抑制作用；然而，它们对弹性蛋白酶的抑制作用较弱。添加抑肽酶未能抑制人胰凝乳蛋白酶和弹性蛋白酶。人白蛋白显示出最小的抑制作用，并且仅作为竞争性抑制剂起作用。人白蛋白、胎牛血清或抑肽酶均未观察到对胶原酶的抑制作用。
本研究清楚地表明，在人胰岛分离过程中，少量内源性胰腺酶仍保持活性，并且在分离过程结束时添加的抑制剂不能完全有效地抑制这些酶。
