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目的通过抑制Twist1来观察胰腺癌细胞对吉西他滨化疗敏感性的变化。方法通过靶向人Twist1基因小干扰RNA(siRNA)抑制Twist1的表达;用Western法检测Twist1的表达、Annexin V/PI染色和流式细胞术检测吉西他滨诱导的细胞凋亡;通过Western blot检测caspase及可能的丝裂原活化蛋白激酶(MAPK)和线粒体途径;用激光扫描共聚焦显微镜检测细胞内活性氧和线粒体膜的电位。结果 Twist1沉默显著增加了Panc-1细胞对吉西他滨的敏感性,JNK/线粒体的途径被激活,Twist1 siRNA诱导线粒体膜去极化同时显著上调线粒体分裂相关蛋白Fis1并降低融合相关蛋白Mfn1的表达。Twist1 siRNA提高了细胞内活性氧(ROS)的水平,与吉西他滨联合治疗进一步增加ROS。结论 siRNA介导的Twist1沉默在PANC-1细胞对吉西他滨化疗耐受中扮演了关键角色,Twist1沉默可能作为胰腺癌治疗的一种有效方法。
Objective To observe the changes of gemcitabine chemosensitivity in pancreatic cancer cells by inhibiting Twist1. Methods Twist1 expression was inhibited by targeting human Twist1 siRNA. Twist1 expression was detected by Western blot. Gemcitabine-induced apoptosis was detected by Annexin V / PI staining and flow cytometry. Caspase-3 Possible mitogen-activated protein kinase (MAPK) and mitochondrial pathway; the potential of intracellular reactive oxygen species and mitochondrial membrane was detected by laser scanning confocal microscopy. Results Twist1 silencing significantly increased the sensitivity of Panc-1 cells to gemcitabine. The JNK / mitochondrial pathway was activated. Twist1 siRNA induced mitochondrial membrane depolarization and up-regulated mitochondrial Fis1 and decreased the expression of Mfn1. Twist1 siRNA increased intracellular levels of reactive oxygen species (ROS) and combined with gemcitabine increased ROS further. Conclusion siRNA-mediated silencing of Twist1 plays a key role in the tolerance of gemcitabine to PANC-1 cells. Twist1 silencing may be an effective method for the treatment of pancreatic cancer.