Facoetti Angelica, Mariotti Luca, Ballarini Francesca, Bertolotti Alessia, Nano Rosanna, Pasi Francesca, Ranza Elena, Ottolenghi Andrea
Dipartimento di Fisica Nucleare e Teorica, Universita degli Studi di Pavia, Pavia, Italy.
Int J Radiat Biol. 2009 Aug;85(8):690-9. doi: 10.1080/09553000903020016.
To clarify the experimental conditions that might influence the release of cytokines in the culture medium and give some basic input for building a model for cytokine (e.g., Interleukin-6, IL-6) regulation in the case of 'sham irradiation' and after ionising radiation exposure.
The influence of cell type, cell density, medium volume, medium storage temperature and other methodological aspects on IL-6 and Interleukin-8 (IL-8) release were investigated. In addition, the effects over the time of different doses of gamma irradiation on the clonogenic survival of bystander cells and on the secretion of these cytokines were studied.
We observed significant decreases of clonogenic survival in AG01522 and T98G cells after the transfer of medium collected 5 and 20 h after low doses of gamma irradiation. Concerning the Interleukins' measurements, our experiments showed that the aggregate removal modalities tested, and up to 10 freeze-thaw cycles, do not have significant influence on the measurements of IL-6 concentration in the medium. We also observed that the IL-6 accumulated in the medium of human fibroblasts is not degraded when maintained at 37 degrees C. Sets of experiments demonstrated that cell density or medium volume do not influence the release of IL-6. On the contrary, our results showed that IL-8 released by glioblastoma cells strongly depends on the amount of medium. Finally, the exposure of fibroblasts to gamma irradiation has influence on the release kinetics of both IL-6 and IL-8 with peculiar features.
This study solved some of the methodological doubts concerning the study of bystander effects by means of the medium transfer technique; moreover it also highlighted some experimental aspects that need to be considered when approaching this sort of experiments.
阐明可能影响培养基中细胞因子释放的实验条件，并为构建“假照射”和电离辐射暴露情况下细胞因子（如白细胞介素-6，IL-6）调控模型提供一些基础数据。
研究了细胞类型、细胞密度、培养基体积、培养基储存温度及其他方法学因素对IL-6和白细胞介素-8（IL-8）释放的影响。此外，还研究了不同剂量γ射线照射对旁观者细胞克隆形成存活率及这些细胞因子分泌的时间效应。
我们观察到，低剂量γ射线照射后5小时和20小时收集的培养基转移后，AG01522和T98G细胞的克隆形成存活率显著降低。关于白细胞介素的测量，我们的实验表明，所测试的聚集去除方式以及多达10次冻融循环对培养基中IL-6浓度的测量没有显著影响。我们还观察到，人成纤维细胞培养基中积累的IL-6在37℃下保存时不会降解。一系列实验表明，细胞密度或培养基体积不会影响IL-6的释放。相反，我们的结果表明，胶质母细胞瘤细胞释放的IL-8强烈依赖于培养基的量。最后，成纤维细胞暴露于γ射线照射对IL-6和IL-8的释放动力学有独特影响。
本研究解决了通过培养基转移技术研究旁观者效应时的一些方法学疑问；此外，还突出了进行这类实验时需要考虑的一些实验方面。
