Wolters G H, Vos-Scheperkeuter G H, van Deijnen J H, van Schilfgaarde R
Department of Surgery, University of Groningen, The Netherlands.
Diabetologia. 1992 Aug;35(8):735-42. doi: 10.1007/BF00429093.
Crude Clostridium histolyticum collagenase is widely used for the enzymatic degradation of pancreatic extracellular matrix in order to isolate the islets of Langerhans. The variable enzymatic composition of crude collagenases is a critical issue which contributes to the poor reproducibility of islet isolation procedures. In this study, the separate contributions of collagenase and protease to the islet isolation process were analysed by testing various combinations of purified collagenase and purified protease in rat pancreas dissociations under conditions which eliminated all other proteolytic activity. Under these conditions, complete tissue dissociation by purified collagenase required 99 +/- 10 min, whereas increasing amounts of protease progressively reduced this time to a minimum of 36 +/- 1 min. Histochemical analysis of the dissociation process showed that protease enhanced the degradation of all four major components of the extracellular matrix: collagen was degraded more completely, while proteoglycans, glycoproteins and elastin were degraded at a higher rate. Pancreas dissociation under the present, strictly controlled conditions resulted in a high yield of viable islets: 4.2-5.0 microliters islet tissue volume (3,300-3,800 islets) were isolated per g pancreas in the presence of a high or low protease concentration, respectively. Prolonged dissociation in the presence of protease resulted in a dramatic decrease in islet yield which correlated with the observation that the enzyme accelerated islet disintegration. It is concluded that the collagenase-induced dissociation of the extracellular matrix is facilitated by protease. Our study shows that high yields of viable islets can be obtained under controlled enzymatic conditions, provided that the exposure of islets to protease is limited.
粗制溶组织梭菌胶原酶被广泛用于酶解胰腺细胞外基质，以分离胰岛。粗制胶原酶的酶组成各异，这是一个关键问题，导致胰岛分离程序的可重复性较差。在本研究中，通过在消除所有其他蛋白水解活性的条件下，测试纯化胶原酶和纯化蛋白酶的各种组合对大鼠胰腺进行解离，分析了胶原酶和蛋白酶对胰岛分离过程的单独作用。在这些条件下，纯化胶原酶完全解离组织需要99±10分钟，而蛋白酶量的增加逐渐将此时间缩短至最短36±1分钟。对解离过程的组织化学分析表明，蛋白酶增强了细胞外基质所有四种主要成分的降解：胶原降解更彻底，而蛋白聚糖、糖蛋白和弹性蛋白降解速度更快。在目前严格控制的条件下进行胰腺解离，可获得高产率的活胰岛：在高或低蛋白酶浓度下，每克胰腺分别分离出4.2 - 5.0微升胰岛组织体积（3300 - 3800个胰岛）。在蛋白酶存在下长时间解离导致胰岛产量急剧下降，这与该酶加速胰岛解体的观察结果相关。得出的结论是，蛋白酶促进了胶原酶诱导的细胞外基质解离。我们的研究表明，在受控的酶条件下可以获得高产率的活胰岛，前提是胰岛暴露于蛋白酶的时间有限。
