Rose Natisha L, Palcic Monica M, Shapiro A M James, Lakey Jonathan R T
Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Centre, University of Alberta, Edmonton, Alberta, T6G 2N8, Canada.
Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada.
Cell Transplant. 2004 Mar;13(2):153-160. doi: 10.3727/000000004773301834.
Despite advances in human islet isolation, islet yield remains inconsistent and unreliable. In recent studies, it has been suggested that serine proteases, in particular trypsin, have been shown to have a damaging effect on islet yield. This study evaluated enzyme activity levels throughout 42 human islet isolation procedures. Trypsin, chymotrypsin, and elastase activity was determined spectrophotometrically using suitable chromophoric substrates. The results of the islet isolations were rated as successful (n = 19) or unsuccessful (n = 23) based on the islet yield and functionality. The enzyme activity profiles of the isolations were compared. No significant differences in donor-related variables were found in this study. However, in the successful isolations, a significantly greater amount (85.6 ± 1.9%; p = 0.0017) of the pancreas was digested in a significantly shorter digestion time (19.7 ± 0.6 min; p = 0.0054) compared with 74.8 ± 2.5% of digested tissue in 22.6 ± 0.7 min in the poor isolations. This study showed no significant effect of serine protease levels on the outcome of islet isolations, regardless of enzyme inhibitor supplementation. These data suggest that serine protease activity does not sufficiently affect islet yield. However, the data show that the most successful human islet isolations are achieved when the maximum amount of tissue is digested in the shortest amount of time. This suggests that further understanding of the isolation process should focus on the role of the collagenase digestion solution in the dissociation of the endocrine-exocrine tissue connection.
尽管人类胰岛分离技术取得了进展，但胰岛产量仍然不稳定且不可靠。在最近的研究中，有人提出丝氨酸蛋白酶，特别是胰蛋白酶，已被证明对胰岛产量有破坏作用。本研究评估了42例人类胰岛分离过程中的酶活性水平。使用合适的发色底物通过分光光度法测定胰蛋白酶、糜蛋白酶和弹性蛋白酶的活性。根据胰岛产量和功能，将胰岛分离的结果评为成功（n = 19）或失败（n = 23）。比较了分离过程中的酶活性谱。本研究未发现供体相关变量存在显著差异。然而，与不成功的分离相比，成功的分离中，胰腺消化量显著更多（85.6 ± 1.9%；p = 0.0017），消化时间显著更短（19.7 ± 0.6分钟；p = 0.0054），而不成功的分离中消化组织为74.8 ± 2.5%，消化时间为22.6 ± 0.7分钟。本研究表明，无论是否补充酶抑制剂，丝氨酸蛋白酶水平对胰岛分离结果均无显著影响。这些数据表明丝氨酸蛋白酶活性对胰岛产量的影响不足。然而，数据显示，在最短时间内消化最大量组织时，人类胰岛分离最为成功。这表明，对分离过程的进一步理解应聚焦于胶原酶消化液在内分泌 - 外分泌组织连接解离中的作用。
