细胞分裂间期和分裂期ERK活性研究

ERK activity in cellular interphase and mitosis

【目的】研究分裂间期和分裂期细胞外信号调节蛋白激酶(ERK)活性,以期了解细胞的生理活动。【方法】培养COS7、Hela-H2B和Cho-EGFR细胞,用有或无诺考达唑(Nocodazole)处理后获得分裂期和分裂间期细胞;再用表皮生长因子(EGF)或AG1478处理,分别收集分裂期和分裂间期细胞,裂解后制备蛋白样品,SDS-PAGE后进行免疫杂交,检查蛋白的表达情况。【结果】细胞经EGF刺激后,EGFR在分裂间期和分裂期出现明显磷酸化。MAPK通路下游蛋白ERK磷酸化水平在分裂间期显著高于分裂期(P<0.05)。EGF质量浓度可影响EGFR的活性,但对ERK磷酸化并未产生明显的影响;在相同条件下,1 ng/mL EGF处理的ERK磷酸化水平与其他质量浓度的EGF处理差异不显著(P>0.05)。0.5μmol/L AG1478可明显抑制EGFR活性,并导致Hela-H2B细胞的ERK磷酸化水平降低;而AG1478浓度为2.5μmol/L时,COS7细胞的ERK磷酸化才明显被抑制。【结论】EGF介导的ERK磷酸化水平在细胞分裂间期高于分裂期;抑制EGFR活性后可影响ERK磷酸化,但这种作用存在细胞差异性。

【Objective】 Through the investigation of ERK activity in cellular interphase and mitosis in response to EGF,the cellular physical characteristic were measured.【Method】 COS7,Hela-H2B and Cho-EGFR cells were cultured.After cells were treated with or without nocodazole,interphase cells and mitotic cells were obtained.Then treated with or without EGF and AG1478,the cells were collected and lysated respectively.The protein expression was detected through SDS-PAGE and immunoblotting.【Result】 After the cells were stimulated with EGF,EGFR phosphorylation in cell interphase and mitosis was increased.And it was found that ERK phosphorylation in cell interphase was higher than that in cell mitosis（P0.05）.EGFR phosphorylation was affected by the dosage of EGF stimulation but ERK phosphorylation almost not.And under the same condition,ERK phophorylation in response to 1 ng/mL EGF was not significant compared with that in response to other concentration of EGF（P0.05）.EGFR activity was significantly inhibited by 0.5 μmol/L AG1478.Next the results showed that ERK phosphorylation of Hela-H2B cells was inhibited in response to 0.5 μmol/L AG1478.Nevertheless,only 2.5 μmol/L was the concentration of AG1478,ERK phosphorylation in COS7 cells was significantly inhibited.【Conclusion】 EGF-mediated ERK phosphorylation in cell interphase is higher than that in cell mitosis.Down-stream ERK phosphorylation are affected after EGFR activity is inhibited,the results of which are different in different cells.