Kin Tatsuya, O'Gorman Doug, Zhai Xiaojun, Pawlick Rena, Imes Sharleen, Senior Peter, Shapiro A M James
Clinical Islet Transplant Program, University of Alberta and Alberta Health Services, Edmonton, Alberta, Canada.
Transplantation. 2009 Jun 15;87(11):1700-5. doi: 10.1097/TP.0b013e3181a60240.
Successful islet isolation relies heavily on enzyme products. Among them, Liberase was used in islet transplantation programs until the islet community was notified of the use of a bovine brain component during the manufacturing process. To minimize potential risk of prion disease transmission, many islet isolation facilities switched to Serva enzyme, which is considered to pose less risk. However, this conversion significantly affected the field in transplant activity. Here, we report our successful conversion from Liberase to Serva collagenase with the use of a modified digestion protocol.
We compared the quality of Serva versus Liberase enzyme using chromatography and collagenase activity assay. On the basis of the findings, we developed a pancreas digestion protocol optimized for Serva enzyme, where only collagenase was injected into the pancreas through the duct, and then neutral protease was added to the circulating system during the digestion phase.
Class I collagenase activity of Serva was remarkably reduced compared with Liberase. Chromatography of Serva demonstrated suspected degradation of class I collagenase. When the modified protocol was applied to donor pancreata more than 35 years of age, we recovered 3119+/-147 islet equivalent/g pancreas with a success rate of 51% (35/68), whereas the standard method yielded only 1809+/-266 islet equivalent/g pancreas (P=0.02) with a success rate of 11% (1/9). This beneficial effect of the modified method was, however, diminished when applied to younger donor pancreata.
Our study brings new insight into the role of collagenase and noncollagenolytic protease on pancreas dissociation.
成功的胰岛分离在很大程度上依赖于酶制品。其中，在胰岛移植项目中一直使用 Liberase，直到胰岛界得知其在生产过程中使用了牛脑成分。为了将朊病毒疾病传播的潜在风险降至最低，许多胰岛分离机构改用了 Serva 酶，认为其风险较小。然而，这种转变对移植活动领域产生了重大影响。在此，我们报告通过使用改良的消化方案，成功地从 Liberase 转换为 Serva 胶原酶。
我们使用色谱法和胶原酶活性测定法比较了 Serva 酶与 Liberase 酶的质量。基于这些发现，我们制定了一种针对 Serva 酶优化的胰腺消化方案，即仅通过导管将胶原酶注入胰腺，然后在消化阶段向循环系统中添加中性蛋白酶。
与 Liberase 相比，Serva 的 I 类胶原酶活性显著降低。Serva 的色谱分析显示 I 类胶原酶疑似降解。当将改良方案应用于 35 岁以上的供体胰腺时，我们每克胰腺回收了 3119±147 个胰岛当量，成功率为 51%（35/68），而标准方法每克胰腺仅产生 1809±266 个胰岛当量（P=0.02），成功率为 11%（1/9）。然而，当应用于较年轻的供体胰腺时，改良方法的这种有益效果减弱。
我们的研究为胶原酶和非胶原olytic 蛋白酶在胰腺解离中的作用带来了新的见解。
