Abdelli Saida, Papas Klearchos K, Mueller Kate R, Murtaugh Mike P, Hering Bernhard J, Bonny Christophe
Departement of Medical Genetics, Centre Hospitalier Universitaire Vaudois (CHUV) and University of Lausanne, Lausanne, Switzerland.
Department of Surgery, University of Arizona, Institute for Cellular Transplantation, Tucson, Arizona, United States of America.
PLoS One. 2014 Jul 1;9(7):e99796. doi: 10.1371/journal.pone.0099796. eCollection 2014.
Stress conditions generated throughout pancreatic islet processing initiate the activation of pro-inflammatory pathways and beta-cell destruction. Our goal is to identify relevant and preferably beta-specific markers to assess the activation of beta-cell stress and apoptotic mechanisms, and therefore the general quality of the islet preparation prior to transplantation. Protein expression and activation were analyzed by Western blotting and kinase assays. ATP measurements were performed by a luminescence-based assay. Oxygen consumption rate (OCR) was measured based on standard protocols using fiber optic sensors. Total RNA was used for gene expression analyzes. Our results indicate that pancreas digestion initiates a potent stress response in the islets by activating two stress kinases, c-Jun N-terminal Kinase (JNK) and p38. JNK1 protein levels remained unchanged between different islet preparations and following culture. In contrast, levels of JNK3 increased after islet culture, but varied markedly, with a subset of preparations bearing low JNK3 expression. The observed changes in JNK3 protein content strongly correlated with OCR measurements as determined by the Spearman's rank correlation coefficient rho [Formula: see text] in the matching islet samples, while inversely correlating with c-fos mRNA expression [Formula: see text]. In conclusion, pancreas digestion recruits JNK and p38 kinases that are known to participate to beta-cell apoptosis. Concomitantly, the islet isolation alters JNK3 and c-fos expression, both strongly correlating with OCR. Thus, a comparative analysis of JNK3 and c-fos expression before and after culture may provide for novel markers to assess islet quality prior to transplantation. JNK3 has the advantage over all other proposed markers to be islet-specific, and thus to provide for a marker independent of non-beta cell contamination.
在整个胰岛处理过程中产生的应激条件会引发促炎途径的激活和β细胞的破坏。我们的目标是识别相关的、最好是β细胞特异性的标志物，以评估β细胞应激和凋亡机制的激活情况，从而评估移植前胰岛制备的总体质量。通过蛋白质印迹法和激酶测定法分析蛋白质表达和激活情况。通过基于发光的测定法进行ATP测量。使用光纤传感器根据标准方案测量氧消耗率（OCR）。总RNA用于基因表达分析。我们的结果表明，胰腺消化通过激活两种应激激酶——c-Jun氨基末端激酶（JNK）和p38，在胰岛中引发强烈的应激反应。在不同的胰岛制备物之间以及培养后，JNK1蛋白水平保持不变。相比之下，胰岛培养后JNK3的水平升高，但变化显著，一部分制备物的JNK3表达较低。在匹配的胰岛样本中，通过Spearman等级相关系数rho [公式：见原文]确定，观察到的JNK3蛋白含量变化与OCR测量值密切相关，而与c-fos mRNA表达呈负相关[公式：见原文]。总之，胰腺消化会募集已知参与β细胞凋亡的JNK和p38激酶。同时，胰岛分离会改变JNK3和c-fos的表达，二者均与OCR密切相关。因此，培养前后JNK3和c-fos表达的比较分析可能会提供新的标志物，用于评估移植前的胰岛质量。JNK3相对于所有其他提议的标志物具有优势，它是胰岛特异性的，因此可以提供一个独立于非β细胞污染的标志物。
