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目的探讨Akt和p27蛋白控制胆管癌细胞生长的分子机制。方法利用突变载体、突变失活载体以及位点突变基因的转染技术,流式细胞仪检测PI3K、转染各类载体对胆管癌细胞周期的影响,免疫印迹、免疫荧光检测PI3K抑制剂、转染各类载体对P27蛋白表达及分布的影响。结果 PI3K抑制剂LY294002引起胆管癌细胞株G1期细胞周期阻滞,并引起p27蛋白入核;Akt能通过诱导p27蛋白出细胞核而解除LY294002的G1期细胞周期阻滞;Akt通过磷酸化野生型p27蛋白第157位苏氨酸逆转细胞周期阻滞。结论 Akt通过磷酸化p27第157位苏氨酸来诱导p27细胞内重定位于细胞质,促进胆管癌细胞的生长。
Objective To investigate the molecular mechanism of Akt and p27 protein in the control of cholangiocarcinoma cell growth. Methods The transfected PI3K cells were transfected with mutated vector, mutated inactivated vector and site-directed mutagenesis gene. The effect of PI3K transfected with various vectors on the cell cycle of cholangiocarcinoma was detected by Western blot. The PI3K inhibitor was detected by immunofluorescence Effect of Various Vectors on P27 Protein Expression and Distribution. Results PI3K inhibitor LY294002 caused cell cycle arrest in G1 phase of bile duct carcinoma cell line and caused p27 protein to enter the nucleus. Akt could release G1 phase cell cycle of LY294002 by inducing p27 out of nucleus. Akt phosphorylated wild-type p27 Protein 157th threonine reverses cell cycle arrest. Conclusion Akt induces the intracellular repression of p27 intracellular cytoplasm by phosphorylating threonine 157 of p27 and promotes the growth of cholangiocarcinoma cells.