Rose Natisha L, Palcic Monica M, Lakey Jonathan R T
Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada.
Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Centre, University of Alberta, Edmonton, Alberta, T6G 2N8, Canada.
Cell Transplant. 2002 Nov;11(8):821-826. doi: 10.3727/000000002783985279.
Inconsistencies in human islet yields after collagenase digestion have been attributed to the activation of endogenous enzymes of the donor pancreas. It has been suggested that pancreatic serine proteases contribute to the proteolysis of collagenase. This study defined the effects of endogenous enzymes within the pancreas on pancreas dissociation during collagenase digestion. Levels of collagenase activity from samples taken throughout several steps in islet isolation procedures, both with and without the addition of the serine protease inhibitor Pefabloc, were determined by a spectrophotometric assay using N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala as the substrate. Results clearly demonstrated that the level of collagenase activity remains stable throughout the isolation procedure despite differences in the donor factors from several cadaveric donor pancreases. This was further demonstrated by observing no difference in activity levels after incubating commercial collagenase preparations with serine proteases and analyzing by means of collagenase activity and SDS-PAGE. These data show that the presence of serine proteases does not affect the level of collagenase activity; however, they likely damage the islet cells upon prolonged digestion of the pancreatic tissue. Further efforts at examining exogenous and endogenous enzyme levels may result in the development of an enzyme cocktail that is both stable and effective for digesting the human pancreas while preserving islet function and viability.
胶原酶消化后人胰岛产量的不一致归因于供体胰腺内源性酶的激活。有人提出胰腺丝氨酸蛋白酶会导致胶原酶的蛋白水解。本研究确定了胰腺内源性酶在胶原酶消化过程中对胰腺解离的影响。在胰岛分离程序的几个步骤中，无论是否添加丝氨酸蛋白酶抑制剂苯甲磺酰氟，采集的样本中的胶原酶活性水平，均通过以N-[3-(2-呋喃基)丙烯酰基]-亮氨酸-甘氨酸-脯氨酸-丙氨酸为底物的分光光度法测定。结果清楚地表明，尽管来自几个尸体供体胰腺的供体因素存在差异，但在整个分离过程中胶原酶活性水平保持稳定。将商业胶原酶制剂与丝氨酸蛋白酶一起孵育，然后通过胶原酶活性和SDS-PAGE分析，结果进一步表明活性水平没有差异。这些数据表明，丝氨酸蛋白酶的存在不会影响胶原酶活性水平；然而，在胰腺组织长时间消化后，它们可能会损害胰岛细胞。进一步研究外源性和内源性酶水平，可能会开发出一种酶混合物，既能稳定有效地消化人胰腺，又能保持胰岛功能和活力。
