1Division of Advanced Surgical Science and Technology, Tohoku University School of Medicine, Sendai, Japan. 2New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan. 3Graduate school of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo, Japan. 4Graduate school of Biomedical Engineering, Tohoku University, Sendai, Japan. 5Department of Integrative Genomics, Tohoku Medical Megabank Organization (ToMMO), Tohoku University School of Medicine, Sendai, Japan. 6Department of Biochemistry, Center for Regulatory Epigenome and Diseases, Tohoku University Graduated School of Medicine, Sendai, Japan.
Transplantation. 2015 Jul;99(7):1349-55. doi: 10.1097/TP.0000000000000662.
Islet isolation currently requires collagenase, neutral protease and other components. Thermolysin (TL) from Bacillus thermoproteolyticus is the gold standard neutral protease. However, we speculated that neutral protease derived from Clostridium histolyticum (Ch; ChNP) would be biologically superior for islet isolation. Tryptic-like activity has also been reported to be important. Therefore, we focused on clostripain (CP), since it is one of the main proteases in Clostridium histolyticum which possesses tryptic-like activity. We then examined the synergistic effects of highly purified ChNP and CP on rat islet isolation.
The same amount of collagenase was used in all four groups (TL, ChNP, TL+CP and ChNP+CP; n = 12/group). The efficiency was evaluated by the islet yield and function. An immunohistochemical analysis, in vitro digestion assay for each enzyme component and evaluation of the activation of endogenous exocrine proteases during islet isolation were also performed.
The islet yield of the TL group was significantly higher than that of the ChNP group (P < 0.01). The islet yield was dose dependently increased in the ChNP+CP group, but was decreased in the TL + CP group. The islet yield in the ChNP + CP group was significantly higher than that in the TL group, but their islet function was similar. Different specificities for laminin, especially laminin-511, were observed in the TL, ChNP, and CP groups.
Clostripain had a strong synergistic effect with ChNP, but not with TL. Therefore, ChNP and CP, in combination with collagenase derived from the same bacteria, may effectively increase the isolation efficiency without affecting the quality of islets.
胰岛分离目前需要胶原酶、中性蛋白酶和其他成分。来自嗜热芽孢杆菌的耐热酶（TL）是金标准中性蛋白酶。然而，我们推测来自溶组织梭菌的中性蛋白酶（Ch；ChNP）在胰岛分离方面具有更好的生物学特性。也有报道称胰蛋白酶样活性很重要。因此，我们专注于梭菌蛋白酶（CP），因为它是具有胰蛋白酶样活性的溶组织梭菌的主要蛋白酶之一。然后，我们研究了高度纯化的 ChNP 和 CP 对大鼠胰岛分离的协同作用。
所有四组（TL、ChNP、TL+CP 和 ChNP+CP；每组 n = 12）均使用相同量的胶原酶。通过胰岛产量和功能评估效率。还进行了免疫组织化学分析、每种酶成分的体外消化测定以及胰岛分离过程中内源性外分泌蛋白酶激活的评估。
TL 组的胰岛产量明显高于 ChNP 组（P < 0.01）。ChNP+CP 组的胰岛产量呈剂量依赖性增加，但 TL + CP 组的胰岛产量降低。ChNP+CP 组的胰岛产量明显高于 TL 组，但它们的胰岛功能相似。TL、ChNP 和 CP 组对层粘连蛋白，尤其是层粘连蛋白-511 的特异性不同。
Clostripain 与 ChNP 具有很强的协同作用，但与 TL 没有协同作用。因此，与来自同一种细菌的胶原酶联合使用的 ChNP 和 CP 可能会有效地提高分离效率，而不会影响胰岛的质量。
