4E-BP1在电离辐射诱发DNA损伤应激中的作用及其分子机制研究

发布时间：2018-11-20 19:16

【摘要】：目的研究探讨真核翻译起始因子4E-结合蛋白1(e IF4E-binding protein 1,4E-BP1)在电离辐射致细胞DNA损伤反应中尤其是G2/M检查点和纺锤体结构检查点的功能调节作用及机制。方法肝癌Hep G2细胞模型,经60Coγ射线照射诱发DNA损伤,Western blot检测4E-BP1总蛋白和T37/46位点磷酸化蛋白的表达水平;流式细胞术检测细胞周期阻滞情况;分子克隆法构建敲低4E-BP1表达的sh RNA干扰质粒,采用慢病毒表达系统-脂质体转染法构建4E-BP1稳定敲低的细胞系;细胞计数法绘制细胞生长曲线;磷酸化组蛋白H3/Ser10分子标记物免疫标记结合流式细胞术来检测M期细胞及G2/M检查点功能;使用Nocodazole将对照细胞和4E-BP1敲低细胞同步化于有丝分裂的前中期,随后去除Nocodazole,细胞进入G1期,来观察敲低4E-BP1蛋白表达对纺锤体结构检查点和细胞有丝分裂进程的影响。结果Hep G2细胞在4Gy射线照射后4h、8h,4E-BP1的T37/46位点磷酸化水平升高,4E-BP1总蛋白表达含量基本不变,同时细胞周期监测显示4E-BP1磷酸化与细胞发生G2/M期阻滞过程相关联,高峰是发生在照射后10~14h。敲低4E-BP1后,细胞的生长速度变慢,对0.5Gy、1Gy较低剂量照射的敏感性增加;4Gy 60Coγ射线照射后两细胞系的G2/M检查点均激活,但G2期阻滞的解除时间存在差别,照射后12h,转染对照质粒的He La-shvector细胞的M期细胞比例为2.24%而转染敲低4E-BP1蛋白质粒的He La-sh4E-BP1细胞的M期细胞已上升到4.26%。PI3K激酶抑制剂降低4E-BP1蛋白的稳定性。结论4E-BP1的T37/46位点磷酸化蛋白在60Coγ射线的诱导下表达上调,敲低4E-BP1蛋白后细胞生长活力下降,对1Gy及以下的低剂量照射的敏感性增加,不影响G2/M检测点的激活,但4E-BP1蛋白的低表达会引起细胞更快的退出电离辐射引起的G2/M期阻滞,4E-BP1蛋白稳定性受PI3K激酶家族成员调控。
[Abstract]:Objective to investigate the function and mechanism of eukaryotic translation initiation factor 4E- binding protein-1 (e IF4E-binding protein 1 + 4E-BP1) in the response to DNA damage induced by ionizing radiation, especially at G 2 / M checkpoint and spindle structure checkpoint. Methods the expression levels of total 4E-BP1 protein and phosphorylated protein at T37 / 46 site were detected by, Western blot in Hep G2 cell model induced by 60Co 纬 -ray irradiation, cell cycle arrest was detected by flow cytometry, and the expression of 4E-BP1 total protein and phosphorylated protein at T37 / 46 site were detected by flow cytometry. The sh RNA interference plasmid with low 4E-BP1 expression was constructed by molecular cloning, the cell line of 4E-BP1 stable knockout was constructed by lentivirus expression system-liposome transfection, the cell growth curve was drawn by cell count method. The phosphorylated histone H3/Ser10 molecular marker was used to detect the function of M phase cells and G 2 / M checkpoint by flow cytometry. Nocodazole was used to synchronize control cells and 4E-BP1 knockout cells in the first and middle stages of mitosis and then remove Nocodazole, cells into G1 phase to observe the effect of low 4E-BP1 protein expression on spindle structural checkpoint and cell mitosis process. Results the phosphorylation level at T37 / 46 of 4E-BP1 was increased in Hep G2 cells at 4 h after 4Gy irradiation, and the expression of total 4E-BP1 protein was almost unchanged. At the same time, cell cycle monitoring showed that 4E-BP1 phosphorylation was associated with G _ 2 / M phase arrest, and the peak occurred at 10 ~ 14 h after irradiation. When 4E-BP1 was knocked down, the growth rate of the cells became slower, and the sensitivity of the cells to 0.5 Gy / 1 Gy irradiation was higher than that of low dose irradiation. The G2 / M checkpoint of the two cell lines was activated after 4Gy 60Co 纬 -irradiation, but the release time of G2 phase arrest was different. The proportion of M-phase cells in He La-shvector cells transfected with control plasmid was 2.24%, while that of He La-sh4E-BP1 cells transfected with low 4E-BP1 protein particles increased to 4E-. Stability of BP1 protein. Conclusion the expression of phosphorylated protein at site T37 / 46 of 4E-BP1 was up-regulated by 60Co 纬 -rays, and the cell growth activity decreased after knocking down the 4E-BP1 protein, and the sensitivity to low dose irradiation of 1Gy and below was increased, and the activation of G _ 2 / M detection point was not affected. However, the low expression of 4E-BP1 protein resulted in faster withdrawal of cells from G _ 2 / M phase arrest induced by ionizing radiation, and the stability of 4E-BP1 protein was regulated by members of PI3K kinase family.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R818

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