抑制IRE1-XBP1通路增强帕比司他的抗食管鳞癌活性研究
发布时间：2018-10-30 15:20
【摘要】：实验目的:观察帕比司他单用及其联合STF083010对食管癌细胞生长增殖的影响,并初步探讨其作用机制。实验方法:采用CCK8法检测药物对食管癌细胞的生长抑制作用;PI单染法检测细胞周期;Annexin V/PI双染色法检测细胞凋亡;Western blot和Quantitative RT-PCR检测药物作用细胞后蛋白及基因表达水平的变化;采用激光共聚焦法检测药物作用细胞后对内质网形态的影响;构建裸鼠移植瘤模型检测药物在体内抗食管癌的效果。实验结果:帕比司他有效抑制对传统化疗药物不敏感的Kyse450和Kyse510食管癌细胞的生长。帕比司他通过增加组蛋白H3和α-微管蛋白乙酰化水平,上调p21和降低c-myc的表达而诱导细胞凋亡和G2/M期周期阻滞。同时,帕比司他诱导细胞产生内质网应激,激活IRE1-Xbp1信号通路。帕比司他与IRE1核酸内切酶抑制剂STF083010联用显著抑制食管癌细胞增殖,大部分两药联合作用指数(CI)1。与单用帕比司他治疗组相比,帕比司他与STF083010联用组显著抑制IRE1-Xbp1促存活信号通路,抑制依赖HDAC6的聚集小体降解途径,上调内质网应激凋亡标志物DDIT3基因的表达,进而下调Bcl-xL/Bax的比例,激活caspase级联反应,增强PARP切割,从而诱导细胞凋亡。在裸鼠移植瘤模型中,STF083010显著增强帕比司他介导的肿瘤生长抑制及肿瘤细胞坏死的数量。结论:IRE1核酸内切酶抑制剂STF083010在体内外都能够显著促进帕比司他对食管癌增殖的抑制作用,并且促进帕比司他诱导的食管癌细胞发生凋亡。
[Abstract]:Aim: to observe the effect of pabestat alone and its combination with STF083010 on the growth and proliferation of esophageal cancer cells and to explore its mechanism. Methods: CCK8 assay was used to detect the growth inhibition of esophageal cancer cells, PI single staining method was used to detect cell cycle, and Annexin V/PI double staining method was used to detect apoptosis. Western blot and Quantitative RT-PCR were used to detect the changes of protein and gene expression, and laser confocal method was used to detect the morphology of endoplasmic reticulum (ER). A nude mouse model of transplanted tumor was established to detect the effect of drugs on esophageal cancer in vivo. Results: Papillast effectively inhibited the growth of Kyse450 and Kyse510 esophageal cancer cells insensitive to traditional chemotherapeutic agents. Pabestar induces apoptosis and G 2 / M cycle arrest by increasing the acetylation level of histone H3 and 伪 -tubulin, up-regulating p21 and decreasing the expression of c-myc. At the same time, paracetamil induces endoplasmic reticulum stress and activates IRE1-Xbp1 signaling pathway. The combination of pabestat and IRE1 endonuclease inhibitor STF083010 significantly inhibited the proliferation of esophageal cancer cells. The combined action index of most of the two drugs was (CI) 1. Compared with the control group, the combination of paracetamol and STF083010 significantly inhibited the IRE1-Xbp1 survival signaling pathway, inhibited the aggregation body degradation pathway dependent on HDAC6, and up-regulated the expression of DDIT3 gene, the endoplasmic reticulum stress marker of apoptosis. Furthermore, the ratio of Bcl-xL/Bax was down-regulated, caspase cascade was activated, PARP cleavage was enhanced, and apoptosis was induced. In nude mice model of transplanted tumor, STF083010 significantly enhanced the amount of tumor growth inhibition and tumor cell necrosis mediated by paracetamil. Conclusion: IRE1 endonuclease inhibitor STF083010 can significantly inhibit the proliferation of esophageal carcinoma and induce apoptosis of esophageal cancer cells induced by pabestatin in vitro and in vivo.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1
[Abstract]:Aim: to observe the effect of pabestat alone and its combination with STF083010 on the growth and proliferation of esophageal cancer cells and to explore its mechanism. Methods: CCK8 assay was used to detect the growth inhibition of esophageal cancer cells, PI single staining method was used to detect cell cycle, and Annexin V/PI double staining method was used to detect apoptosis. Western blot and Quantitative RT-PCR were used to detect the changes of protein and gene expression, and laser confocal method was used to detect the morphology of endoplasmic reticulum (ER). A nude mouse model of transplanted tumor was established to detect the effect of drugs on esophageal cancer in vivo. Results: Papillast effectively inhibited the growth of Kyse450 and Kyse510 esophageal cancer cells insensitive to traditional chemotherapeutic agents. Pabestar induces apoptosis and G 2 / M cycle arrest by increasing the acetylation level of histone H3 and 伪 -tubulin, up-regulating p21 and decreasing the expression of c-myc. At the same time, paracetamil induces endoplasmic reticulum stress and activates IRE1-Xbp1 signaling pathway. The combination of pabestat and IRE1 endonuclease inhibitor STF083010 significantly inhibited the proliferation of esophageal cancer cells. The combined action index of most of the two drugs was (CI) 1. Compared with the control group, the combination of paracetamol and STF083010 significantly inhibited the IRE1-Xbp1 survival signaling pathway, inhibited the aggregation body degradation pathway dependent on HDAC6, and up-regulated the expression of DDIT3 gene, the endoplasmic reticulum stress marker of apoptosis. Furthermore, the ratio of Bcl-xL/Bax was down-regulated, caspase cascade was activated, PARP cleavage was enhanced, and apoptosis was induced. In nude mice model of transplanted tumor, STF083010 significantly enhanced the amount of tumor growth inhibition and tumor cell necrosis mediated by paracetamil. Conclusion: IRE1 endonuclease inhibitor STF083010 can significantly inhibit the proliferation of esophageal carcinoma and induce apoptosis of esophageal cancer cells induced by pabestatin in vitro and in vivo.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1
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