Rödel Franz, Schaller Ulrich, Schultze-Mosgau Stefan, Beuscher Horst-Ulrich, Keilholz Ludwig, Herrmann Martin, Voll Reinhard, Sauer Rolf, Hildebrandt Guido
Department of Radiooncology, University of Erlangen-Nuremberg, Erlangen, Germany.
Strahlenther Onkol. 2004 Apr;180(4):194-200. doi: 10.1007/s00066-004-1237-y.
Low-dose radiotherapy (LD-RT) is known to exert an anti-inflammatory effect, but the knowledge of the underlying molecular mechanisms is still scarce. The authors have recently reported that transforming growth factor beta 1 (TGF-beta(1)) essentially contributes to a reduced endothelial adhesion of mononuclear cells (PBMC) following LD-RT. Furthermore, TGF-beta(1) secretion was associated with the induction of the transcription factor nuclear factor kappa B (NF-kappaB). However, the time course of adhesion, TGF-beta(1) expression, and NF-kappaB activity following LD-RT has not been thoroughly investigated yet.
The human EA.hy.926 endothelial cell line (EA.hy.926 EC) was grown to 95% confluence. Immediately after stimulation with the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha), EA.hy.926 EC were irradiated with single doses ranging from 0.3 up to 3 Gy. Adhesion assays were performed 4, 12, and 24 h after irradiation. Nuclear extracts and culture supernatants were harvested after 4, 8, 12, 20, 24, 30, and 40 h. NF-kappaB DNA-binding activity was analyzed by electrophoretic mobility shift assays (EMSA) and TGF-beta(1) secretion by enzyme-linked immunosorbent assay (ELISA). The functional impact of TGF-beta(1) on the course of leukocyte/EC adhesion was analyzed by neutralizing TGF-beta(1) in parallel with stimulation/irradiation of the EA.hy.926 EC.
4 and 24 h after irradiation of EA.hy.926 EC in the dose range between 0.3 and 0.7 Gy, a reduced adhesion of PBMC compared to nontreated controls could be observed. However, 12 h after irradiation a relative maximum of adhesion (up to 30% increase) was seen at a dose of 0.3 Gy. TGF-beta(1) secretion and NF-kappaB DNA-binding activity displayed a similar biphasic kinetics of induction with a relative minimum 12 h after irradiation. Neutralization of TGF-beta(1) activity restored adhesion at 4 and 24 h after LD-RT of EA.hy.926 EC, but it did not influence leukocyte adhesion 12 h after irradiation.
LD-RT of stimulated human EA.hy.926 EC is followed by a biphasic time course of NF-kappaB activity and an increased secretion of TGF-beta(1). The kinetics shows peak levels at 4-8 h and 24-30 h after LD-RT and results in a biphasic leukocyte/EC adhesion profile.
已知低剂量放疗（LD-RT）具有抗炎作用，但对其潜在分子机制的了解仍然有限。作者最近报道，转化生长因子β1（TGF-β(1)）在低剂量放疗后对单核细胞（PBMC）与内皮细胞的黏附减少起着重要作用。此外，TGF-β(1)的分泌与转录因子核因子κB（NF-κB）的诱导有关。然而，低剂量放疗后黏附、TGF-β(1)表达和NF-κB活性的时间进程尚未得到充分研究。
将人EA.hy.926内皮细胞系（EA.hy.926 EC）培养至95%汇合。在用促炎细胞因子肿瘤坏死因子α（TNF-α）刺激后，立即用0.3至3 Gy的单剂量对EA.hy.926 EC进行照射。在照射后4、12和24小时进行黏附试验。在照射后4、8、12、20、24、30和40小时收集细胞核提取物和培养上清液。通过电泳迁移率变动分析（EMSA）分析NF-κB DNA结合活性，通过酶联免疫吸附测定（ELISA）分析TGF-β(1)分泌。通过在刺激/照射EA.hy.926 EC的同时中和TGF-β(1)，分析TGF-β(1)对白细胞/内皮细胞黏附过程的功能影响。
在0.3至0.7 Gy剂量范围内照射EA.hy.926 EC后4和24小时，与未处理的对照组相比，可观察到PBMC黏附减少。然而，照射后12小时，在0.3 Gy剂量下观察到相对最大的黏附（增加高达30%）。TGF-β(1)分泌和NF-κB DNA结合活性显示出相似的双相诱导动力学，照射后12小时相对最低。中和TGF-β(1)活性可恢复EA.hy.926 EC低剂量放疗后4和24小时的黏附，但不影响照射后12小时的白细胞黏附。
受刺激的人EA.hy.926 EC进行低剂量放疗后，NF-κB活性呈双相时间进程，TGF-β(1)分泌增加。动力学显示在低剂量放疗后4 - 8小时和24 - 30小时达到峰值水平，并导致双相白细胞/内皮细胞黏附谱。
