查尔酮衍生物cc-85调节活性氧诱导食管癌细胞凋亡的作用机制研究

发布时间：2017-12-31 03:16

本文关键词：查尔酮衍生物cc-85调节活性氧诱导食管癌细胞凋亡的作用机制研究　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的主要研究新型查尔酮类衍生物cc-85对食管癌细胞系的体外作用机制。以Nrf2为靶点,通过死亡受体途径和线粒体途径,研究cc-85的抗肿瘤作用机理,为肿瘤药物的开发提供理论依据。方法通过一系列药理学及分子生物学实验方法研究课题内容,主要包括MTT法检测细胞活力、流式细胞术检测活性氧水平和线粒体膜电位、蛋白免疫印迹法检测蛋白表达水平、DAPI法检测细胞核形态和免疫荧光法检测蛋白定位等。结果Cc-85抑制食管癌细胞株EC109,KYSE750和EC-1的细胞生存率,结果表明cc-85对正常的食管上皮细胞毒性相对较小,而对EC109和KYSE750较为敏感,抑制率显著;cc-85能够诱导EC109和KYSE750细胞的凋亡;DAPI染色表明EC109细胞核形态变小、染色加深并且出现凋亡小体;cc-85明显上调EC109细胞核KYSE750细胞内外源性受体DR4和DR5的表达;cc-85可以诱导线粒体膜电位下降,细胞内抗凋亡蛋白和促凋亡蛋白表达水平的变化;caspase-3,8,9可以被cc-85诱导活化,caspase的全酶抑制剂z-VAD-fmk可以部分阻断化合物cc-85诱导EC109的凋亡;ROS抑制剂NAC可以逆转cc-85诱导的EC109和KYSE750细胞的凋亡;NAC可以逆转cc-85诱导的EC109细胞的线粒体膜电位水平;NAC可以逆转cc-85诱导的EC109细胞的抗凋亡蛋白和促凋亡蛋白的表达水平;NAC可以逆转cc-85诱导的EC109细胞的caspase家族的蛋白表达;cc-85可以诱导Nrf2定位于细胞核。结论以上结果说明查尔酮衍生物cc-85可以诱导食管癌细胞的凋亡。Cc-85可以通过Nrf2的入核转录、死亡受体DR4和DR5的增高、线粒体膜电位的下降、抗凋亡蛋白和促凋亡蛋白的调节等一系列信号诱导凋亡,可以作为抗肿瘤药物开发的重要理论依据。
[Abstract]:Objective to study the mechanism of action of cc-85, a novel chalcone derivative, on esophageal cancer cell line in vitro. Nrf2 was used as the target, through death receptor pathway and mitochondrial pathway. To study the anti-tumor mechanism of cc-85 and to provide theoretical basis for the development of tumor drugs. Methods A series of pharmacological and molecular biological experimental methods were used to study the subject. MTT assay was used to detect cell viability, flow cytometry to detect reactive oxygen species (Ros) and mitochondrial membrane potential (MMP), and Western blot to detect protein expression. Results Cc-85 inhibited the survival rate of esophageal cancer cell line EC109 KYSE750 and EC-1. The results showed that the toxicity of cc-85 to normal esophageal epithelial cells was relatively small, but sensitive to EC109 and KYSE750, and the inhibition rate was significant. Cc-85 could induce apoptosis of EC109 and KYSE750 cells. DAPI staining showed that the nuclear morphology of EC109 became smaller, the staining deepened and apoptotic bodies appeared. Cc-85 upregulated the expression of DR4 and DR5 in EC109 nuclear KYSE750 cells. Cc-85 could induce the decrease of mitochondrial membrane potential and the expression of anti-apoptotic protein and pro-apoptotic protein. Caspase-3, caspase-3 and caspase-8 can be activated by cc-85. Z-VAD-fmk, a holoenzyme inhibitor of caspase, partially blocked the apoptosis of EC109 induced by cc-85. ROS inhibitor NAC could reverse the apoptosis of EC109 and KYSE750 cells induced by cc-85. NAC could reverse the mitochondrial membrane potential level of EC109 cells induced by cc-85. NAC could reverse the expression of anti-apoptotic protein and pro-apoptotic protein in EC109 cells induced by cc-85. NAC could reverse the protein expression of caspase family in EC109 cells induced by cc-85. Conclusion the above results suggest that cc-85 can induce apoptosis of esophageal carcinoma cells. Cc-85 can be transcripted by Nrf2. . The increase of death receptor DR4 and DR5, the decrease of mitochondrial membrane potential, the regulation of anti-apoptotic protein and pro-apoptotic protein signal induce apoptosis, which can be used as an important theoretical basis for the development of anti-tumor drugs.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

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