Effect of GR knockdown and GSH depletion on the in vitro interaction between B16 melanoma cells and the vascular endothelium.

HSE cells (2.5×105cells/well) cultured for 24 h were co-cultured with B16-F10 or iB16-shGCR cells (5.0×105cells/well; pre-cultured for 24 h). Twenty minutes after the addition of tumor cells to the HSE, the plates were washed as described in Materials and Methods. The ratio of tumor cells adhering to the HSE was 1∶1. TNF-α (100 units/ml) and IFN-γ (50 units/ml), which were used as potent activators of NO and H2O2 generation by the HSE, were added to the co-cultures when all tumor cells present were attached to the HSE. In endothelium-induced B16-F10/iB16-shGCR cytotoxicity assays, tumor cytotoxicity (expressed as the % of tumor cells that lost viability within the 3–6-h incubation period) was determined after 6 h of incubation. During the 6-h incubation period, the percentage of HSE cell viability was 98–99% in all cases. When adding cytokines to cultured tumor cells alone, no cytostatic or cytotoxic effects were observed within the next 6 h. During the first 2-hincubation period, both HSE and B16-F10 or iB16-shGCR cells maintained >95% viability (data not shown). Where indicated, B16-F10 or iB16-shGCR cells were incubated for 24 h with BSO (0.5 mM) before co-culturing with endothelial cells. Pretreatment of B16-F10 cells with BSO did not significantly affect control values for tumor cell adhesion. Data are means ± S.D. for 5–6 independent experiments. *pversus B16-F10 + HSE controls in the absence of BSO.将培养24小时的肝窦内皮细胞（HSE，2.5×10⁵个/孔）与预培养24小时的B16-F10或iB16-shGCR肿瘤细胞（5.0×10⁵个/孔）进行共培养。向HSE培养体系中加入肿瘤细胞20分钟后，依照《材料与方法》所述流程洗涤培养板。黏附于HSE的肿瘤细胞与HSE的数量比例为1∶1。肿瘤坏死因子-α（TNF-α，100单位/ml）与干扰素-γ（IFN-γ，50单位/ml）作为可有效诱导HSE产生一氧化氮（NO）与过氧化氢（H₂O₂）的激活剂，在所有肿瘤细胞均黏附于HSE后，被加入共培养体系中。在内皮细胞介导的B16-F10/iB16-shGCR细胞毒性实验中，于孵育6小时后检测肿瘤细胞毒性（以3~6小时孵育期内丧失活力的肿瘤细胞百分比表示）。在6小时的孵育期间，所有实验组的HSE细胞活力均维持在98%~99%。若仅向单独培养的肿瘤细胞中加入细胞因子，则在后续6小时内未观察到任何细胞生长抑制与细胞毒性效应。在最初2小时的孵育期内，HSE、B16-F10及iB16-shGCR细胞的活力均维持在95%以上（数据未展示）。如实验标注所示，在与内皮细胞共培养前，将B16-F10或iB16-shGCR细胞与0.5 mM的丁硫氨酸亚砜胺（BSO）共同孵育24小时。用BSO预处理B16-F10细胞，不会显著影响肿瘤细胞黏附的对照组数值。实验数据以5~6次独立实验的均值±标准偏差（S.D.）呈现。*代表与无BSO存在时的B16-F10+HSE对照组相比具有统计学差异。