Centre Européen d'Etude du Diabète, Strasbourg, France.
Cell Transplant. 2012;21(4):657-69. doi: 10.3727/096368911X593136. Epub 2011 Sep 22.
As oxygen carriers, perfluorocarbon emulsions might be useful to decrease hypoxia of pancreatic islets before transplantation. However, their hydrophobicity prevents their homogenisation in culture medium. To increase the surface of contact between islets and Perfluorooctyl bromide (PFOB), and consequently oxygen delivery, we tested effect of a PFOB emulsion in culture medium on β-cell lines and rat pancreatic islets. RINm5F β-cell line or pancreatic rat islets were incubated for 3 days in the presence of PFOB emulsion in media (3.5% w/v). Preoxygenation of the medium was performed before culture. Cell viability was assessed by apoptotic markers (Bax and Bcl-2) and by staining (fluoresceine diacetate and propidium iodide). β-Cell functionality was determined by insulin release during a glucose stimulation test and. Hypoxia markers, HIF-1α and VEGF, were studied at days 1 and 3 using RT-PCR, Western blotting, and ELISA. PFOB emulsions preserved viability and functionality of RINm5F cells with a decrease of HIF-1α and VEGF expression. Islets viability was preserved during 3 days of culture. Secretion of VEGF was higher in untreated control (0.09 ± 0.041 μg VEGF/mg total protein) than in PFOB emulsion incubated islets (0.02 ± 0.19 μg VEGF/mg total protein, n = 4, p < 0.05) at day 1. At day 3, VEGF secretion was increased as compared to day 1 in control (0.23 ± 0.04 μg VEGF/mg total protein) but it was imbalance by the presence of PFOB emulsion (0.09 ± 0.03 μg VEGF/mg total protein, n = 5, p < 0.05). While insulin secretion was maintained in response to a glucose stimulation test until day 3 when islets were incubated in the presence of PFOB emulsion preoxygenated (0.81 ± 0.16 at day 1 vs. 0.75 ± 0.24 at day 3), the ability to secrete insulin in the presence of high glucose concentration was lost in islets controls (0.51 ± 0.18 at day 1 vs. 0.21 ± 0.13 at day 3). Atmospheric oxygen delivery by PFOB emulsion might be sufficient to decrease islets hypoxia. However, to improve islets functionality, overoxygenation is needed. Finally, maintenance of islet viability and functionality for several days after isolation could improve the outcome of islets transplantation.
作为氧载体，全氟碳乳液在移植前降低胰岛缺氧可能是有用的。然而，其疏水性阻止了其在培养基中的均匀化。为了增加胰岛与全氟辛基溴化物（PFOB）之间的接触表面积，并因此增加氧输送，我们测试了 PFOB 乳液在培养基中对β细胞系和大鼠胰岛的影响。RINm5Fβ细胞系或大鼠胰岛在存在 PFOB 乳液的培养基中孵育 3 天（3.5%w/v）。在培养前进行培养基预充氧。通过凋亡标志物（Bax 和 Bcl-2）和染色（荧光素二乙酸酯和碘化丙啶）评估细胞活力。在葡萄糖刺激试验期间测定β细胞功能，并测定胰岛素释放。在第 1 天和第 3 天使用 RT-PCR、Western blot 和 ELISA 研究缺氧标志物 HIF-1α 和 VEGF。PFOB 乳液在降低 HIF-1α 和 VEGF 表达的情况下保持 RINm5F 细胞的活力和功能。在 3 天的培养过程中，胰岛的活力得以维持。与未经处理的对照（0.09±0.041μg VEGF/mg 总蛋白）相比，未孵育 PFOB 乳液的胰岛中 VEGF 的分泌更高（0.02±0.19μg VEGF/mg 总蛋白，n=4，p<0.05）在第 1 天。第 3 天，与第 1 天相比，对照组中的 VEGF 分泌增加（0.23±0.04μg VEGF/mg 总蛋白），但存在 PFOB 乳液会使其失衡（0.09±0.03μg VEGF/mg 总蛋白，n=5，p<0.05）。当胰岛在预充氧的 PFOB 乳液存在下孵育时，胰岛素分泌在第 3 天仍能维持对葡萄糖刺激试验的反应（第 1 天为 0.81±0.16，第 3 天为 0.75±0.24），而对照组中的胰岛在高葡萄糖浓度下分泌胰岛素的能力丧失（第 1 天为 0.51±0.18，第 3 天为 0.21±0.13）。PFOB 乳液提供的大气氧输送可能足以降低胰岛缺氧。然而，为了改善胰岛功能，需要过度供氧。最后，在分离后几天内维持胰岛的活力和功能可以改善胰岛移植的结果。
