Department of Surgery, University of California Irvine, Irvine, California, United States of America.
Department of Materials Science and Engineering, Sue and Bill Gross Stem Cell Research Center, University of California Irvine, Irvine, California, United States of America.
PLoS One. 2020 Dec 7;15(12):e0243506. doi: 10.1371/journal.pone.0243506. eCollection 2020.
Previous studies have shown that necrostatin-1 (Nec-1) supplementation improved the viability of murine islets following exposure to nitric oxide, increased the survival of human islets during hypoxic culture, and augmented the maturation of pre-weaned porcine islets (PPIs) after 7 days of tissue culture. A limitation of these studies is that only one concentration of Nec-1 was used, and no studies have determined the optimal dose of Nec-1 for PPIs. Thus, the present study examined the effects of Nec-1 on PPIs at four different doses-0, 25, 50, 100, and 200 μM-after 7 days of tissue culture when supplemented on day 3. PPIs were isolated from pancreata of pre-weaned Yorkshire piglets (8-15 days old) and cultured in a specific islet maturation media added with Nec-1 on day 3 of tissue culture at 4 different doses-0, 25, 50, 100, and 200 μM (n = 6 for each dose). After 7 days of tissue culture, islets were assessed for recovery, viability, endocrine cellular content, GLUT2 expression in beta cells, and insulin secretion after glucose challenge. Nec-1 did not affect the viability of both intact islets and dissociated islets cells during tissue culture regardless of doses. Islets cultured in media supplemented with Nec-1 at 100 μM, but not 25, 50, or 200 μM, had a significantly higher recovery, composition of endocrine cells, GLUT2 expression in beta cells, and insulin secretion capacity than control islets cultured in media without Nec-1 supplementation. Moreover, culturing islets in 200 μM Nec-1 supplemented media not only failed to improve the insulin release but resulted in a lower glucose-induced insulin stimulation index compared to islets cultured in media added with 100 μM Nec-1. Xenotransplantation using porcine islets continues to demonstrate scientific advances to justify this area of research. Our findings indicate that Nec-1 supplementation at 100 μM was most effective to enhance the in vitro maturation of PPIs during tissue culture.
先前的研究表明，坏死抑制蛋白-1（Nec-1）的补充可以提高在一氧化氮暴露下的鼠胰岛的存活率，增加在低氧培养时人胰岛的存活率，并促进在组织培养 7 天后的未成熟猪胰岛（PPIs）的成熟。这些研究的局限性在于仅使用了一种浓度的 Nec-1，并且没有研究确定 Nec-1 对 PPIs 的最佳剂量。因此，本研究在组织培养第 7 天（添加第 3 天）时，用四种不同浓度（0、25、50、100 和 200μM）的 Nec-1 处理，观察了 Nec-1 对 PPIs 的影响。PPIs 是从 8-15 天大的约克夏仔猪的胰腺中分离出来的，在添加有 Nec-1 的特定胰岛成熟培养基中进行培养，在组织培养的第 3 天用四种不同浓度（0、25、50、100 和 200μM）（每组剂量 n = 6）添加 Nec-1。在组织培养 7 天后，评估胰岛的恢复率、活力、内分泌细胞含量、β细胞中的 GLUT2 表达和葡萄糖刺激后的胰岛素分泌。无论剂量如何，Nec-1 在组织培养过程中均不影响完整胰岛和分离胰岛细胞的活力。在添加 100μM Nec-1 的培养基中培养的胰岛，与不添加 Nec-1 补充剂的培养基中培养的胰岛相比，胰岛的恢复率、内分泌细胞组成、β细胞中的 GLUT2 表达和胰岛素分泌能力显著更高。此外，与在添加 100μM Nec-1 的培养基中培养的胰岛相比，在添加 200μM Nec-1 的培养基中培养的胰岛不仅未能提高胰岛素释放，而且导致葡萄糖诱导的胰岛素刺激指数降低。使用猪胰岛的异种移植继续证明了科学进步，证明了这一研究领域的合理性。我们的研究结果表明，在组织培养过程中，添加 100μM Nec-1 是增强 PPIs 体外成熟的最有效方法。
