ATF5蛋白对食管鳞癌细胞耐药性影响

发布时间：2018-06-27 14:39

  本文选题：AFT蛋白 + 食管鳞癌细胞　； 参考：《中华肿瘤防治杂志》2017年05期

【摘要】：目的食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)多药耐药严重影响化疗效果,其分子机制仍未阐明。本研究通过构建不同ATF5蛋白表达量的食管癌Eca-109细胞模型,探究过表达ATF5及低表达ATF5对食管癌Eca-109细胞耐药性及凋亡的影响。方法利用脂质体载体转染技术对食管癌细胞株进行转染,建立高、中(转染空质粒的对照组)、低3组不同ATF5蛋白表达量Eca-109细胞模型。蛋白质印迹法检测3组细胞ATF5蛋白表达水平。MTT实验和平板克隆实验观察3组细胞对紫杉醇、顺铂耐受性的变化;DAPI染料核染色后观察3组细胞在紫杉醇、顺铂作用下凋亡反应的差异;分析ATF5蛋白对食管鳞癌Eca-109细胞耐药的影响。结果利用转染技术成功构建的3组不同ATF5蛋白表达量的Eca-109细胞模型,表达模型组细胞中ATF5蛋白相对表达量为85.9±2.66,对照组为64.35±2.54,低表达模型组为45.3±3.11,3组差异有统计学意义,F=532.323,P0.001。MTT实验显示,紫杉醇、顺铂作用72h后,3组细胞存活率差异有统计学意义,F_紫=163.382,P0.001;F_顺=579.36,P0.001;同一组细胞不同浓度对存活率的影响差异有统计学意义,F_紫=616.32,P0.001;F_顺=2 558.05,P0.001;不同浓度的紫杉醇、顺铂作用3组细胞后,过表达组细胞的半数抑制率浓度(IC50)分别为4.16±2.21和1.54±0.67;对照组分别为1.54±0.92和1.27±0.65;低表达组细胞的半数抑制率浓度分别为0.33±0.21和0.53±0.62,3组差异有统计学意义,F_紫=198 330.768,P0.001;F_顺=64 298.048,P0.001;表明过表达ATF5的细胞对紫杉醇、顺铂的药物耐受性均明显升高而低表达模型组细胞对紫杉醇、顺铂的药物耐受性均明显降低。DAPI染色实验显示,紫杉醇、顺铂作用3组细胞后,过表达组细胞的凋亡率分别为(14.04±1.66)%和(11.75±2.09)%;对照组分别为(26.44±2.99)%和(34.07±3.42)%;低表达组细胞的凋亡率分别为(54.85±5.88)%和(66.66±2.81)%,3组差异有统计学意义,F_紫=283.976,P0.001;F_顺=954.464,P0.001。提示过表达ATF5蛋白细胞在紫杉醇、顺铂作用下凋亡细胞均减少,低表达ATF5细胞在紫杉醇、顺铂作用下凋亡细胞均增加。平板克隆形成实验显示,紫杉醇、顺铂作用3组细胞后,过表达组细胞的克隆形成率分别为(56.09±1.37)%和(62.67±1.41)%;对照组分别为(38.7±1.37)%和(34.83±3.09)%;低表达组细胞的克隆形成率分别为(25.22±1.58)%和(18.17±3.64)%,3组差异有统计学意义,F_紫=1157.447,P0.001;F_顺=612.595,P0.001。表明过表达ATF5蛋白细胞在紫杉醇、顺铂作用下克隆形成数均更多,低表达ATF5蛋白细胞在紫杉醇、顺铂作用下克隆形成数减少。结论上调ATF5蛋白的表达能增加食管鳞癌Eca-109细胞对紫杉醇、顺铂的耐药性,而下调ATF5蛋白的表达则能降低Eca-109细胞对紫杉醇、顺铂的耐药性,提示食管鳞癌细胞的耐药性可能与ATF5蛋白的表达量相关,食管鳞癌细胞中ATF5蛋白的表达情况可能能间接干扰临床药物化疗的效果。
[Abstract]:Objective multidrug resistance (MDR) in esophageal squamous cell carcinoma (esophageal squamous cell) has a serious effect on the effect of chemotherapy, but its molecular mechanism is still unknown. This study investigated the effects of overexpression of ATF5 and low expression of ATF5 on drug resistance and apoptosis of esophageal carcinoma Eca-109 cells. Methods Eca-109 cells were transfected with liposome vector to establish Eca-109 cell model with high, middle (blank plasmid control group) and low ATF5 protein expression. The expression of ATF5 protein in three groups was detected by Western blotting. MTT assay and plate cloning assay were used to observe the changes of paclitaxel and cisplatin tolerance in the three groups. After DAPI dye nucleus staining, the three groups of cells were observed in paclitaxel. The effect of ATF5 protein on drug resistance of esophageal squamous cell carcinoma Eca-109 cells was analyzed. Results three groups of Eca-109 cell models with different ATF5 expression levels were successfully constructed by transfection technique. The relative expression of ATF5 protein was 85.9 卤2.66 in the model group, 64.35 卤2.54 in the control group, and 45.3 卤3.11 in the low-expression model group. After 72 hours of treatment with paclitaxel and cisplatin, the cell survival rates of the three groups were significantly different. There were significant differences in cell survival rate between the three groups after treatment with paclitaxel and cisplatin for 72 hours. There were significant differences in the survival rate of the same group of cells treated with different concentrations of paclitaxel, 616.32C, P0.001Fx 25558.05, and different concentrations of paclitaxel. After treated with cisplatin, IC50 was 4.16 卤2.21 and 1.54 卤0.67 in overexpression group, 1.54 卤0.92 and 1.27 卤0.65 in control group, and 0.33 卤0.21 in low-expression group and 0.53 卤0.62ng in low expression group, respectively. ATF5 cells against paclitaxel, The drug tolerance of cisplatin group was significantly increased, while that of low expression model group was significantly decreased. The results of DAPI staining showed that paclitaxel and cisplatin treated three groups of cells. The apoptotic rate of overexpression group was (14.04 卤1.66)% and (11.75 卤2.09), that of control group was (26.44 卤2.99)% and (34.07 卤3.42), and that of low expression group was (54.85 卤5.88)% and (66.66 卤2.81), respectively. The results suggested that the number of apoptotic cells with overexpression of ATF5 protein in paclitaxel and cisplatin was decreased, while the apoptotic cells with low expression of ATF5 were increased under paclitaxel and cisplatin. Plate clone formation assay showed that three groups of cells were treated with paclitaxel and cisplatin. The clone formation rates of overexpression group were (56.09 卤1.37)% and (62.67 卤1.41), those of control group were (38.7 卤1.37)% and (34.83 卤3.09), and those of low expression group were (25.22 卤1.58)% and (18.17 卤3.64), respectively. The results showed that the over-expressed ATF5 protein cells had more clone formation under paclitaxel and cisplatin treatment, while the low expression ATF5 protein cells were reduced in taxol and cisplatin. Conclusion upregulation of ATF5 protein can increase the resistance of Eca-109 cells to paclitaxel and cisplatin, while down-regulation of ATF5 protein expression can decrease the resistance of Eca-109 cells to paclitaxel and cisplatin. The results suggest that the drug resistance of esophageal squamous carcinoma cells may be related to the expression of ATF5 protein, and the expression of ATF5 protein in esophageal squamous cell carcinoma cells may indirectly interfere with the effect of clinical drug chemotherapy.
【作者单位】： 广州军区广州总医院消化内科;广州中医药大学第二临床医学院;
【基金】：国家自然科学基金青年项目(81302164) 广东省自然科学基金(2014A030313595) 广州市科技计划(201607010077)
【分类号】：R735.1
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本文编号：2074247