Abdelli S, Abderrahmani A, Hering B J, Beckmann J S, Bonny C
Service of Medical Genetics, CHUV Hospital, Chemin des Falaises 1, 1011 Lausanne-CHUV, Switzerland.
Diabetologia. 2007 Aug;50(8):1660-9. doi: 10.1007/s00125-007-0704-2. Epub 2007 Jun 9.
AIMS/HYPOTHESIS: The protocols used for the preparation of human pancreatic islets immediately induce a sustained and massive activation of the c-Jun-N-terminal kinase (JNK). JNK, which participates in apoptosis of insulin-secreting cells, is activated by mechanical stresses, as well as by exposure to pro-inflammatory cytokines. Here, we investigated whether the delivery of a protease-resistant JNK inhibitory peptide (D-JNKI) through a protein transduction system during pancreatic digestion might impair JNK signalling throughout the transplantation procedure.
Rat pancreases were treated with D-JNKI through the pancreatic duct and cells then isolated by enzymatic digestion. Protein extracts were prepared to determine JNK activity by kinase assays and total RNA was extracted to measure gene expressions by a Light-Cycler technique. Cell apoptosis rate was determined by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and by scoring cells displaying pycnotic nuclei.
Our data establish that the peptide transduction system used here efficiently transfects islets, allowing for stable in vivo (up to 2 days) transfection of human islets transplanted under the kidney capsule. Further, D-JNKI decreases intracellular JNK signalling during isolation and following cytokine exposure in both human and rat islets, as measured by kinase assays and reduced c-fos expression; D-JNKI also confers protection against apoptosis induced during the rat islet preparation and subsequent to IL-1beta exposure.
CONCLUSIONS/INTERPRETATION: JNK signalling participates in islet isolation- and IL-1beta-induced apoptosis in rat islets. Furthermore, the system we used might be more generally applicable for the persistent blockage (several days) of pro-apoptotic pathways in the transplanted islets; this days-long protection might potentially be an absolute prerequisite to help transplanted islets better survive the first wave of the non-specific inflammatory attack.
目的/假设：用于制备人胰岛的实验方案会立即引发c-Jun氨基末端激酶（JNK）的持续且大量激活。JNK参与胰岛素分泌细胞的凋亡，可被机械应激以及暴露于促炎细胞因子激活。在此，我们研究了在胰腺消化过程中通过蛋白质转导系统递送蛋白酶抗性JNK抑制肽（D-JNKI）是否会在整个移植过程中损害JNK信号传导。
通过胰管用D-JNKI处理大鼠胰腺，然后通过酶消化分离细胞。制备蛋白质提取物以通过激酶测定法确定JNK活性，并提取总RNA以通过Light-Cycler技术测量基因表达。通过末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）测定法并对显示核固缩的细胞进行评分来确定细胞凋亡率。
我们的数据表明，此处使用的肽转导系统可有效转染胰岛，使人胰岛在肾被膜下移植后能在体内稳定转染（长达2天）。此外，通过激酶测定法和降低的c-fos表达测量，D-JNKI在人胰岛和大鼠胰岛分离期间以及细胞因子暴露后均可降低细胞内JNK信号传导；D-JNKI还可保护大鼠胰岛在制备过程中以及暴露于IL-1β后诱导的凋亡。
结论/解读：JNK信号传导参与大鼠胰岛分离和IL-1β诱导的凋亡。此外，我们使用的系统可能更普遍适用于移植胰岛中促凋亡途径的持续阻断（数天）；这种长达数天的保护可能是帮助移植胰岛更好地度过非特异性炎症攻击第一波的绝对先决条件。
