摘要
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 verify the roles of tumor necrosis factor α(TNFα) as a growth factor in FBJ cells growth in serum free medium. Methods Cells viability was detected by tetrazolium assay. Expression of TNFα and activation of extracellular signal-regulated kinase (Erk), p38 were determined by reverse transcriptional polymerase chain reaction or western blotting, respectively. Results TNFα addition to FBJ-LL and FBJ-S1 cells significantly enhanced cell survival in serum free medium, transfection of FBJ-LL and FBJ-S1 cells with TNFα sense cDNA markedly enhanced cell survival in serum free medium. SiRNA of Pknl suppressed TNFα as well as Pknl ,in serum free medium,cell survival of protein kinase N 1 silenced cells was suppressed and increased by treatment of TNFα, stimulation of FBJ-LL cells with TNFα increased phospho- rylation of MAPK-Erk rapidly and significantly, silence of Erk by siRNA resulted in cell death comparing to scrambled transfected cells. TNFα has been proved to be suppressed by ganglioside GDla. In this study,it was also found that TNFα enhanced cell growth in serum free medium was suppressed by the presence of GDla. Conclusions TNFα is responsible for cell survival of FBJ cells in serum free medium through Pknl and MAPK-Erk signal pathways.
出处
《沈阳药科大学学报》
CAS
CSCD
北大核心
2009年第2期145-151,共7页
Journal of Shenyang Pharmaceutical University