摘要
目的本实验旨在探讨结肠癌细胞中磷酸化蛋白激酶B(pAkt)对细胞FLICE抑制蛋白(c-FLIP)基因表达的调控作用。方法取对数生长期的HT-29细胞系,分别采用10nM、20nM和40nM的Akt抑制剂wortmannin对其进行干预,收集不同干预时间点的细胞,运用MTT法检测细胞的增殖抑制情况。采用40nM的wortmannin干预HT-29细胞0、3、6、12、24h后分别收集等量细胞,提取细胞总蛋白及总RNA,采用Westernblot检测pAkt和c-FLIP蛋白表达水平,RT-PCR检测c-FLIP基因的转录水平。结果三个浓度的wortmannin都对HT-29细胞的生长具有抑制作用,随着浓度、时间的递增,其抑制效应逐渐增强(P<0.01或0.05),而对照组未见明显变化(P>0.05)。随着wortmannin的干预时间延长,磷酸化Akt蛋白的表达水平明显降低,同时c-FLIP蛋白的表达水平也随之下降,3h后其表达水平已明显降低。wortmannin干预HT-29细胞3h后,与对照组相比其c-FLIPmRNA表达水平已明显下降到40%,24h后其表达水平下降到25%。结论Akt活化的特异性抑制剂wortmannin可抑制体外培养的结肠癌HT-29细胞的生长,结肠癌中可能存在Akt/c-FLIP信号通路调节c-FLIP基因的表达。
Objective To investigate the effect of phosphorylated protein kinase B （pAkt） on the gene expression of cellular FLICE inhibitory protein （c-FLIP） in colon cancer cells. Methods HT-29 cells were treated with 10nM, 20 nM and 40 nM of specific inhibitor of Akt wortmannin for 0, 3, 6, 12 and 24 h. Cells were harvested and cell viability was estimated using MTT assay. Furthermore, total protein and RNA were extracted from the cells which were treated with 40 nm wortmannin for 0, 3, 6, 12 and 24 h, respectively. Western blot was used to detect the level of pAkt and c-FLIP protein and reverse transcdptase-polymerase chain reaction （RT-PCR） was used to determine transcription of c-FLIP mRNA. Results Wortmannin caused a decrease of cell viability. Western blot revealed a significant decrease of the protein expression of pAkt and c-FLIP in HT-29 cells after 3 hour＇s treatment with wortmannin compared with the control. Meanwhile, the expression of c-FLIP mRNA decreased by 40% and 25% compared with control after 3 hour＇s and 24 hour＇s treatment, respectively. Conclusion The findings suggest that wortrnannin inhibits the growth of HT-29 cells that may depend on an Akt/FLIP pathway.
出处
《现代消化及介入诊疗》
2006年第3期129-132,共4页
Modern Interventional Diagnosis and Treatment in Gastroenterology
