人肝细胞癌Huh7细胞中EP3受体不同亚型的鉴定及相关胞内信号转导通路的研究

The identification of the different subtype of EP3 and the related cell signal transduction pathway in human hepatocellular carcinoma

目的:鉴定人肝细胞癌Huh7细胞中EP3受体不同亚型的类型及其介导的细胞内信号转导途径,探讨EP3对人肝细胞癌发生发展的影响及相关分子机制。方法:用RT-PCR实验检测Huh7细胞中EP3亚型的类型,给予EP3受体激动剂sul-prostone处理后,用WST-8法检测细胞的增殖活性,用酶联免疫法测定细胞内环磷酸腺苷(cAMP)浓度的变化,用Western blot-ting法检测Akt、Erk等蛋白磷酸化水平的变化。结果:RT-PCR结果显示人肝细胞癌Huh7细胞中表达的EP3剪接体类型是EP3(5);WST-8实验结果显示,经10.0μmol/LEP3受体激动剂sulprostone作用24h后,细胞增殖率增加46.2%;酶联免疫法显示sulprostone可促使细胞内cAMP浓度显著增高;Western blotting实验显示,sulprostone作用30min可使Huh7细胞中Akt蛋白磷酸化水平增加27%,而Erk蛋白磷酸化水平降低16.4%。结论:人肝细胞癌Huh7细胞中,表达的EP3亚型类型是EP3(5),EP3受体激动剂sulprostone可以促进Huh7细胞的增殖,通过EP3受体的介导促进胞内cAMP表达水平的增加,其机制可能部分涉及到Akt及Erk信号转导通路。

Objective：To investigate the subtype of the EP3 and the related cell signal transduction pathway in human hepatocelluar carcinoma inorder to explore the molecular mechanism. Methods：The Huh7 ceils were treated with different concertrations of Sulprostone for different times. The expression and splicesome type of EP3 was determined by RT-PCR. The cell viability was detected by WST-8. The level of intracellular cAMP concentration was measured by immunoassay Kit. and Western blott was employed to detect the expression of p-Akt and p-Erk. Results：The spliceosome of EP3 expressed in Huh7 is EP3（5）. After the treatment of 10.0 μmoL/L Sulprostone for 24 hours,the cells growth rate was increased 46.2%, and the level of cAMP in the cells was increased greatly. The re markable increase of Akt phosphorylation and decrease of Erk phosphorylation could be observed by Western blott after the treatment of Sulprostone for 30 min. Conclusion： Our results suggested that the subtype of the EP3-EP3（5） mediated a great increasing of the intracellular concentration of cAMP in Huh7 cells,which is partly related to the Akt and Erk signaling pathway.