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目的研究磷酸酰肌醇-3-激酶(PI3K)通过不同亚型的Akt调节细胞凋亡。方法各种肿瘤细胞种植在96孔或6孔细胞培养皿中,采用免疫沉淀加酶联法测定Akt磷酸化状态及其酶活性。通过免疫沉淀加Werstern印迹法,测定Akt活性。用细胞凋亡实验定量分析Akt抑制剂诱导细胞死亡的效应。结果①各类Akt亚型抑制剂可阻断Akt1或Akt2蛋白序列上的第308位点苏氨酸(pAkt308)和第473位点丝氨酸(pAkt473)磷酸化,并表现出相应的Akt亚型抑制特异性;②抑制磷酸化Akt可明显降低其激活底物GSK3β的能力;③多种检测的肿瘤细胞系表现出PI3K通过Akt调节细胞凋亡程序可能主要和两种Akt亚型(Akt1,Akt2)相关。结论PI3K/Akt信号传导途径涉及细胞凋亡,主要通过PI3K下游基因Akt参与调节,Akt1和Akt2亚型是主要调节因子,只有同时抑制Akt1和Akt2才能启动细胞凋亡程序。
AIM To investigate the role of phosphoinositide 3-kinase (PI3K) in the regulation of apoptosis by Akt in different subtypes. Methods A variety of tumor cells were seeded in 96-well or 6-well cell culture dishes. The phosphorylation state and activity of Akt were determined by immunoprecipitation and enzyme-linked immunosorbent assay. Akt activity was determined by immunoprecipitation plus Werstern blotting. Apoptosis experiments were used to quantitatively analyze the effect of Akt inhibitors on cell death. Results ① All kinds of inhibitors of Akt could block the phosphorylation of threonine (pAkt308) and serine (pAkt473) at position 308 of Akt1 or Akt2 protein sequence and showed the corresponding inhibition of Akt subtype Specificity; (2) inhibition of phosphorylation of Akt can significantly reduce its ability to activate the substrate GSK3β; ③ a variety of tumor cell lines showed PI3K apoptosis through Akt program may be mainly and two Akt subtypes (Akt1, Akt2) Related. Conclusion PI3K / Akt signaling pathway involves in apoptosis, which is mainly regulated by PI3K downstream gene Akt. Akt1 and Akt2 is the main regulatory factor. Only by inhibiting Akt1 and Akt2 can initiate apoptosis program.