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目的研究肿瘤坏死因子(TNF-α)对培养的C6胶质瘤细胞中Src抑制的蛋白激酶C底物(Src-suppressed C Kinase Substrate,SSeCKS)表达的影响。方法根据TNF-α刺激时间与浓度的差异,将培养的C6胶质瘤细胞随机分为TNF-α时间刺激组与浓度刺激组,运用实时荧光定量PCR(Realtime PCR)、免疫印迹和免疫细胞化学法分析SSeCKS的表达变化和亚细胞定位。结果细胞因子TNF-α可引起C6胶质瘤细胞中蛋白激酶C(Protein ki-nase C,PKC)底物的广泛磷酸化,并以时间及浓度依赖的方式上调PKC底物SSeCKS的表达。免疫细胞化学分析显示,正常情况下,SSeCKS散在分布于细胞质,浓集于细胞伸长的足突中。TNF-α刺激后,SSeCKS向核周迁移。这些改变可被PKC的抑制剂Ro-31-8220部分抑制。结论TNF-α可诱导C6胶质瘤细胞中PKC的活性,上调SSeCKS表达,这些改变与PKC 的活性相关,提示SSeCKS可能参与胶质细胞中炎症信号的转导。
Objective To investigate the effect of tumor necrosis factor-α (TNF-α) on the expression of Src-suppressed C kinase inhibitor (Src-KS) in cultured C6 glioma cells. Methods The cultured C6 glioma cells were randomly divided into two groups according to the time and concentration of TNF-α. Real-time PCR, Western blot and immunocytochemistry Analysis of SSeCKS expression changes and subcellular localization. Results The cytokine TNF-α induced broad phosphorylation of protein kinase C (PKC) in C6 glioma cells and up-regulated the expression of PKC substrate SSeCKS in a time and concentration-dependent manner. Immunocytochemistry analysis showed that under normal conditions, SSeCKS scattered in the cytoplasm, concentrated in the cell elongation of the foot process. After the stimulation of TNF-α, SSeCKS migrated to the perinuclear area. These changes can be partially inhibited by PKC inhibitor Ro-31-8220. Conclusion TNF-α can induce the activity of PKC in C6 glioma cells and up-regulate the expression of SSeCKS. These changes are correlated with the activity of PKC, suggesting that SSeCKS may be involved in the transduction of inflammatory signals in glial cells.