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本文采用与各种细胞信号传导相关的抑制剂阻断重组人β肿瘤坏死因子(rhTNF-β)对1929靶细胞的杀伤作用、并配合荧光染色、DNA电泳和FCM等实验手段,检测rhTNF-β)引起L929细胞凋亡及加入抑制剂以后的变化,这为研究TNF-β在杀伤肿瘤细胞过程中信号传导的特征提供了基础资料.hTNF-β为本实验室自行重组并纯化之产品,比活性5×10~5U/mg.信号传导仰制剂包括:Genistein (酪氨酸激酶抑制剂);Indomethacin(磷脂酶抑制剂);Quinacrine(磷脂酶抑制剂);Staurosporine(蛋白激酶 C抑制剂); TMB8(钙桔抗剂和蛋白激酶抑制剂);TPCK(丝氨酸蛋白激酶抑制剂); Verapamil(钙通道阻断剂); Wortmannin(磷脂酶 A2抑制剂)均为Sigma产品.采用:①信号传导阻断试验:将L929细胞加入96孔细胞培养板内,每孔1.5×10~4/0.1 ml/孔,培养12h后换以新鲜培养液,并加入50μl各种抑制剂及50μl rhTNF-β(终浓度62 U/ml),另设 rhTNF-β及1640培养液的对照孔.继续培养48h,用结晶紫染色方法计算存活率.②荧光染色:用以上相同的方法处理1929细胞,然后用PI及FDA双染色,荧光显微镜观察.③DNA电泳:经处理的L929细胞,通过
In this study, we used various inhibitors of cell signaling to block the killing effect of rhTNF-β on target 1929 cells. Fluorescent staining, DNA electrophoresis and FCM were used to detect the effect of rhTNF-β ) Induced the apoptosis of L929 cells and the changes after addition of inhibitors, which provided the basic information for the study of the signal transduction characteristics of TNF-β in killing tumor cells.HTNF-β was the product of self-recombination and purification in our laboratory Activity 5 × 10 ~ 5U / mg. Signal transduction agents include: Genistein (tyrosine kinase inhibitor); Indomethacin (phospholipase inhibitor); Quinacrine (phospholipase inhibitor); Staurosporine (protein kinase C inhibitor); TMB8 (calcium citrate antagonist and protein kinase inhibitor); TPCK (serine protein kinase inhibitor); Verapamil (calcium channel blocker); Wortmannin (phospholipase A2 inhibitor) Broken test: The L929 cells were added into 96-well cell culture plates at a density of 1.5 × 10 ~ 4 / 0.1 ml / well and cultured for 12h, then replaced with fresh medium and 50μl of various inhibitors and 50μl of rhTNF-β Concentration 62 U / ml), another set of rhTNF-β and 1640 medium pairs Continue to grow 48h, calculated by the method of crystal violet staining survival rate.②Fluorescence staining: The same way above treated 1929 cells, and then double staining with PI and FDA, fluorescence microscopy.③DNA electrophoresis: treated L929 cells, by