Effects of rG-CSF on Proliferation of Urological Cancer Cells

用OK-432培养PBMC抑制PBMCs产生rG-CSF。这种抑制作用可能在OK-432的细胞因子介导的抗肿瘤作用机制中发挥作用。在以下实验中，将肿瘤细胞（如KK-47细胞和T24细胞）和PBMCs彼此接种在分开的琼脂层中。培养上清中未检测到IL-6、EGF、bFGF、IL-2等细胞因子。KK-47细胞在上清液中组成性地产生碱性FGF和IL-6，T24细胞产生IL-6。rG-CSF与KK-47细胞共培养96小时后，KK-47细胞碱性FGF的产生量呈剂量依赖性增加，直至rG-CSF浓度达到10 ng/ml。此外，在培养基中存在PBMC和rG-CSF的情况下，碱性FGF的产生增加。当碱性-FGF加入到培养基中时，通过MTT测定法估计的KK-47细胞的细胞数以剂量依赖性方式增加碱性-FGF浓度。rG-CSF和PBMC联合培养的KK-47细胞数明显高于单独培养的KK-47细胞。rG-CSF可使KK-47细胞S期细胞数增加，而T24细胞S期细胞数增加不明显。RT-PCR结果显示，KK-47细胞表达IL-6受体、FGF受体1和rG-CSF受体的mRNA，但未见FGF受体2 mRNA的表达。T24细胞表达IL-6 mRNA，而不表达rG-CSF mRNA。PBMC表达rG-CSF、FGF受体1和2的mRNA。提示IL-6和碱性FGF可能是KK-47细胞的自分泌因子。rG-CSF激活KK-47细胞产生碱性成纤维细胞生长因子可能参与rG-CSF对KK-47细胞增殖的影响。

The cultivation of PBMCs with OK-432 inhibited the production of rG-CSF by PBMCs. The inhibition may play a role in the mechanism of the cytokine-mediated antitumor effect of OK-432.Tumor cells such as KK-47 cells and T24 cells, and PBMCs were seeded in separated agar layr each other in the following experiments. Any cytokines such as IL-6, EGF,basic-FGF,IL-2 were not detected in the supernatant of cultured PBMCs. KK-47 cells constitutively produced basic-FGF and IL-6 in the supernatant and T24 cells produced IL-6. When KK-47 cells were cultured with rG-CSF for 96 hours, productio of basic-FGF by KK-47 cells increased in a dose-dependent manner until rG-CSF concentraion of 10 ng/ml. Furthermore, the production of basic-FGF increased under the presence of both of PBMCs and rG-CSF in the culture medium. When basic-FGF was added into culture medium, cell number estimated by MTT assay of KK-47 cells increased in a dose-dependent manner on basic-FGF concentrations. Cell number of KK-47 cells cultured with both of rG-CSF and PBMCs significantly increased than those cultured with rG-CSF only. Cell number of S-phase fraction estimated by FCM of KK-47 cells increased in cultivation with rG-CSF,however, that of T24 cells did not in the same condition. In RT-PCR,KK-47 cells expressed mRNAs of IL-6 receptor, FGF receptor 1 and rG-CSF receptor, however, no expression of mRNA of FGF receptor 2 was observed. T24 cells expressed mRNA of IL-6, however, no expression of mRNA of rG-CSF was observed. PBMCs expressed mRNAs of rG-CSF,FGF receptor 1 and 2. It was suggested that IL-6 and jbasic FGF might be autocrine factors of KK-47 cells. Production of basic FGF by KK-47 cells activated by rG-CSF might take part in effect of rG-CSF on proliferation of KK-47 cells.