Isoprostanes as Markers of Oxidative Stress-Induced Cell Damage in Porcine Islet Cell Isolation
Isoprostanes as Markers of Oxidative Stress-Induced Cell Damage in Porcine Islet Cell Isolation
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DOI：
10.1016/j.transproceed.2009.11.047
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发表时间：
2010-06-01
影响因子：
0.9
通讯作者：
Stadlbauer, V.
中科院分区：
文献类型：
--
作者：
Stiegler, P.;Schaffellner, S.;Stadlbauer, V.
导论.在分离和纯化过程中使用的机械应力和试剂以及消化时间和温度可以改变猪胰岛细胞（PIC）分离的成功率。本研究旨在使用改良的自动Ricordi方法表征PIC分离过程中异前列烷的发生，并评估其对PIC分离结果的影响。在当地屠宰场收获猪胰腺组织，并使用改良的自动Ricordi方法进行10次PIC分离。作为组织损伤相关氧化应激的阳性对照，在1 μ g脂多糖（LPS）存在下进行6次连续PIC分离。使用Lymphoprep密度梯度通过密度梯度离心纯化PLC。采用酶联免疫吸附法测定异前列烷。实验组中活的和纯的PIC的最终产量为3479 +/-542IEQ/g胰腺，并且与实验组相比，LPS组产生更低的细胞数量。在从消化过程开始的分离期间的所有时间点，与实验组相比，LPS组中的异前列烷水平显著升高。PIC分离和纯化结果在两个实验组中显著不同，强调了氧化应激对PIC活力和纯度的负面影响，这对PIC移植成功产生负面影响。
Introduction. Mechanical stress and reagents used during the isolation and purification process as well as digestion time and temperature can alter the success of porcine islet cell (PIC) isolation. This study aimed to characterize the occurrence of isoprostanes during PIC isolation using a modified automated Ricordi method and to evaluate their influence on PIC isolation outcome.Methods. Porcine pancreatic tissue was harvested at the local slaughter house, and 10 PIC isolations were performed using a modified automated Ricordi method. As positive controls for tissue damage-associated oxidative stress, six consecutive PIC isolations were performed in the presence of 1 mu g lipopolysaccharide (LPS). PLC were purified by density gradient centrifugation using the Lymphoprep density gradient. Isoprostane measurement was performed using enzyme-linked immunosorbent assay.Results. The final yield of viable and pure PICs in the experimental group was 3479 +/- 542 IEQ/g pancreas, and the LPS group yielded lower cell numbers compared to the experimental group. Isoprostane levels were significantly elevated in the LPS group as compared to the experimental group at all time points during the isolation from the beginning of the digestion process.Discussion. PIC isolation and purification results significantly differed in the two experimental groups, underlining the negative effects of oxidative stress on PIC viability and purity, which impact negatively on PIC transplantation success.