2 Gy X射线对EL-4细胞p16、CyclinD、CDK4基因转录和蛋白表达影响

Influence on gene transcription and protein expression of p16,CyclinD,CDK4 of EL-4 cell line after 2Gy X-irradiation

目的 研究电离辐射对EL 4细胞p16、CyclinD、CDK4基因转录及蛋白表达的影响。探讨p16 CyclinD CDK4负向调控通路在辐射诱导G1期阻滞中的作用。方法 采用半定量RT PCR方法检测 2GyX射线照射后EL 4细胞p16mRNA水平变化 ,采用Northernblot方法检测CyclinD、CDK4基因转录变化 ,应用免疫细胞化学方法 (ICC)检测 3种蛋白表达的变化。结果 2GyX射线照射后 2～ 4 8hEL 4细胞p16mRNA水平增高 ,4 8h恢复至正常水平。p16蛋白表达照射后 2h开始增高 ,12h达到峰值 (P <0 0 1) ,72h恢复至正常水平。EL 4细胞周期蛋白CyclinDmRNA水平于照射后 1h开始明显降低 ,照射后 8h降至最低值。CyclinD蛋白表达于照射后 8h开始明显减少 ,持续至照射后4 8h (P <0 0 5～P <0 0 1)。EL 4细胞CDK4mRNA水平于照射后 1h明显降低 ,照射后 4h～ 12h保持较低水平 ,72h恢复至正常水平。CDK4蛋白表达于照射后 4h明显下降 ,12h降至最低 ,72h仍低于正常水平 (P <0 0 1)。结论 2GyX射线照射可诱导EL 4细胞p16表达增高 ,CyclinD和CDK4表达降低。提示p16 CyclinD CDK4负向调控通路可能在电离辐射诱导EL 4细胞G1期阻滞中发挥重要作用。

Objective To investigate the effect of ionizing radiation on p16, CyclinD, CDK4 gene transcription and protein expression of EL-4 cells and to elucidate the role of p16/CyclinD/CDK4 pathways in G1 arrest. Methods RT-PCR method was employed to check p16 mRNA level. Northern blot was employed to determine CyclinD and CDK4 mRNA level. Immunocytochemstry was used to measure their protein expression. Results p16 mRNA level was dramatically increased at 2～48 h, recovered at 48h. p16 protein expression was significantly elevated at 12 h ( P <0.01). CyclinD mRNA level of EL-4 cells was dramatically declined at 1 h, reached the lowest level at 8 h.CyclinD protein expression of EL-4 cells was significantly decreased at 8h and lasted to 48 h (P<0.05～P<0 .01). There was a significantly descent in CDK4 mRNA level of EL-4 cells at 1 h after 2Gy X-irradiation. CDK4 mRNA level remained the lower level during 4 h to 12 h, recovered at 72 h. CDK4 protein expression of EL-4 cells dramatically reduced at 4 h, reached the bottom at 12 h, didnot recover at 72 h ( P< 0.01) after 2Gy X-irradiation. Conclusion The increasing of p16 and the reduction of CyclinD, CDK4 gene transcription and protein expression of EL-4 cells were observed after ionizing radiation. The results suggested that p16/CyclinD/CDK4 negative pathway mat play an important role in G1 arrest of EL-4 cells induced by X-rays irradiation. ;