Fujio Atsushi, Murayama Kazutaka, Yamagata Youhei, Watanabe Kimiko, Imura Takehiro, Inagaki Akiko, Ohbayashi Naomi, Shima Hiroki, Sekiguchi Satoshi, Fujimori Keisei, Igarashi Kazuhiko, Ohuchi Noriaki, Satomi Susumu, Goto Masafumi
Division of Advanced Surgical Science and Technology, Tohoku University, Sendai, Japan.
Cell Transplant. 2014;23(10):1187-98. doi: 10.3727/096368913X668654. Epub 2013 Jun 13.
The role(s) of collagenase G (ColG) and collagenase H (ColH) during pancreatic islet isolation remains controversial, possibly due to the enzyme blends used in the previous studies. We herein examined the role of ColG and ColH using highly pure enzyme blends of recombinant collagenase of each subtype. Rat pancreases were digested using thermolysin, together with ColG, ColH, or ColG/ColH (n = 9, respectively). No tryptic-like activity was detected in any components of the enzyme blends. The efficiency of the collagenase subtypes was evaluated by islet yield and function. Immunohistochemical analysis, in vitro collagen digestion assay, and mass spectrometry were also performed to examine the target matrix components of the crucial collagenase subtype. The islet yield was highest in the ColG/ColH group (4,101 ± 460 islet equivalents). A substantial number of functional islets (2,811 ± 581 islet equivalents) was obtained in the ColH group, whereas no islets were retrieved in the ColG group. Mass spectrometry demonstrated that ColH reacts with collagen I and III. In the immunohistochemical analysis, both collagen I and III were located in exocrine tissues, although collagen III expression was more pronounced. The collagen digestion assay showed that collagen III was more effectively digested by ColH than by ColG. The present study reveals that ColH is crucial, while ColG plays only a supporting role, in rat islet isolation. In addition, collagen III appears to be one of the key targets of ColH.
胶原酶G（ColG）和胶原酶H（ColH）在胰岛分离过程中的作用仍存在争议，这可能是由于先前研究中使用的酶混合物所致。我们在此使用每种亚型的重组胶原酶的高纯度酶混合物来研究ColG和ColH的作用。使用嗜热菌蛋白酶以及ColG、ColH或ColG/ColH消化大鼠胰腺（每组n = 9）。在酶混合物的任何成分中均未检测到类胰蛋白酶活性。通过胰岛产量和功能评估胶原酶亚型的效率。还进行了免疫组织化学分析、体外胶原消化试验和质谱分析，以检查关键胶原酶亚型的靶基质成分。ColG/ColH组的胰岛产量最高（4101±460个胰岛当量）。ColH组获得了大量功能性胰岛（2811±581个胰岛当量），而ColG组未获得任何胰岛。质谱分析表明ColH与I型和III型胶原反应。在免疫组织化学分析中，I型和III型胶原均位于外分泌组织中，尽管III型胶原的表达更为明显。胶原消化试验表明，ColH比ColG更有效地消化III型胶原。本研究表明，在大鼠胰岛分离中，ColH至关重要，而ColG仅起辅助作用。此外，III型胶原似乎是ColH的关键靶标之一。
