Department of Internal Medicine and Therapeutics, University of Pavia.
Eur J Histochem. 2020 Mar 26;64(2):3123. doi: 10.4081/ejh.2020.3123.
Isolated hepatocytes can be obtained from the liver by collagenase infusion, a procedure that could affect cell isolation as well as the integrity of membrane receptors. In this respect we compared metabotropic glutamate subtype 5 receptor (mGluR5) protein expression and activity in rat hepatocytes isolated by two collagenases, type I and type IV. Hepatocytes were isolated from male Wistar rats (200-250 g) using collagenase I or collagenase IV and after isolation, viability and morphology of rat hepatocytes were assessed measuring mGluR5 protein expression by Western blot analyses. mGluR5 activation was evaluated by inositol-1-phosphate (IP-1) accumulation after treatment with the mGluR5 orthosteric agonist ACPD or the selective antagonist MPEP. No difference in cellular viability and morphology was observed using collagenase I when compared with collagenase IV. An increase in mGluR5 protein expression was observed in hepatocytes isolated using collagenase IV with respect to collagenase I. Moreover, hepatocytes treated with ACPD and with MPEP presented higher levels of IP-1 when isolated using collagenase IV compared to collagenase I. These results indicate that collagenase IV better preserves the activity of surface proteins such as mGluR5 in isolated rat hepatocytes, an in vitro model useful to reduce the use of experimental animals, in line with the 3R ethical framework.
可以通过胶原酶输注从肝脏中获得分离的肝细胞，该过程可能会影响细胞分离以及膜受体的完整性。在这方面，我们比较了两种胶原酶（I 型和 IV 型）分离的大鼠肝细胞中代谢型谷氨酸受体 5（mGluR5）蛋白表达和活性。使用胶原酶 I 或胶原酶 IV 从雄性 Wistar 大鼠（200-250g）中分离肝细胞，并在分离后，通过 Western blot 分析测量 mGluR5 蛋白表达来评估大鼠肝细胞的活力和形态。通过用 mGluR5 正构激动剂 ACPD 或选择性拮抗剂 MPEP 处理来评估 mGluR5 的激活。与胶原酶 IV 相比，使用胶原酶 I 时细胞活力和形态没有差异。与胶原酶 I 相比，使用胶原酶 IV 分离的肝细胞中 mGluR5 蛋白表达增加。此外，用 ACPD 和 MPEP 处理的肝细胞在使用胶原酶 IV 分离时比使用胶原酶 I 分离时具有更高水平的 IP-1。这些结果表明，胶原酶 IV 可更好地保持分离的大鼠肝细胞表面蛋白（如 mGluR5）的活性，这是一种有用的体外模型，可减少实验动物的使用，符合 3R 伦理框架。
