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目的研究肿瘤坏死因子α(tumor necrosis factorα,TNFα)在无血清条件下作为生长因子促进FBJ细胞生长的信号通路。方法用细胞活性检测法分析细胞的活性;分别用逆转录聚合酶链式反应(reverse transcriptional-polymerase chain reaction,RT-PCR)法和免疫印迹法检测细胞中TNFα的表达以及胞外信号调节激酶(extracellular signal-regulated kinase,Erk)、p38有丝分裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)的磷酸化水平。结果在无血清培养基培养条件下,经TNFα(10μg.L-1)处理的FBJ细胞明显对抗无血清条件下诱发的细胞死亡,TNFαsense cD-NA转染的FBJ-LL和FBJ-S1细胞均能提高其在正常培养基以及无血清培养基中的生长速度,TNFα是FBJ细胞中重要的生长因子之一;蛋白酶N1(protein kinase N1,Pkn1)siRNA可明显沉默目的基因Pkn1的表达,同时降低TNFα的含量;Pkn1沉默的细胞在无血清条件下的生长明显被抑制;TNFα处理的细胞能迅速刺激Erk的磷酸化,Erk沉默的FBJ-LL细胞的生长速度明显降低,TNFα通过Erk信号通路诱发细胞生长;TNFα亦能诱导另一有丝分裂原活化蛋白激酶(mitogen-ac-tivated protein kinase,MAPK)家族p38的磷酸化;神经节苷脂GD1a可降低TNFα在FBJ细胞中的表达,TNFα诱发的无血清条件下的生长速度也被神经节苷脂GD1a抑制。结论在无血清条件下,TNFα通过Pkn1和MAPK-Erk通路调控小鼠骨肉瘤FBJ细胞的生长。
Objective To investigate the role of tumor necrosis factor α (TNFα) as a signaling pathway for the growth of FBJ cells under serum-free conditions. Methods Cell viability assay was used to analyze the activity of the cells. Reverse transcriptional polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression of TNFα and extracellular signal-regulated kinase in cells. The phosphorylation levels of extracellular signal-regulated kinase (Erk) and p38 mitogen-activated protein kinase (p38 MAPK). Results The FBJ cells treated with TNFα (10μg.L-1) were significantly resistant to cell death induced by serum-free conditions in serum-free medium, and both FBJ-LL and FBJ-S1 cells transfected with TNFαsense cD-NA were It can increase the growth rate in normal culture medium and serum-free culture medium. TNFα is one of the important growth factors in FBJ cells; protein kinase N1 (Pkn1) siRNA can significantly silence the expression of the target gene Pkn1 and reduce it. TNFα content; Pkn1-silenced cells were significantly inhibited from growth under serum-free conditions; TNFα-treated cells rapidly stimulated Erk phosphorylation, and Erk-silenced FBJ-LL cells showed a significantly lower growth rate, and TNFα was induced via the Erk signaling pathway. Cell growth; TNFα can also induce the phosphorylation of another mitogen-ac-tivated protein kinase (MAPK) family p38; ganglioside GD1a can reduce TNFα expression in FBJ cells, TNFα induced no The growth rate under serum conditions was also inhibited by ganglioside GD1a. Conclusions Under serum-free conditions, TNFα regulates the growth of bone marrow sarcoma FBJ cells through Pkn1 and MAPK-Erk pathways.