Gli抑制剂GANT61对食管鳞癌细胞OE21凋亡及侵袭能力的影响

发布时间：2018-01-26 08:43

本文关键词： 食管鳞癌细胞凋亡侵袭转移 Gli1 GANT61　出处：《河北医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的:食管癌是我国常见的消化道肿瘤之一,我国食管癌患者的发病率和死亡率均居世界首位,其病理类型以食管鳞状细胞癌为主。目前食管癌的手术、放化疗治疗效果欠佳,患者生存预后不理想,其分子靶向领域的治疗越来越得到人们的重视。Hedgehog(Hh)信号通路在多细胞生物胚胎发育过程中发挥关键性作用。Hh信号通路的活化参与成体生物组织的干细胞调控、组织损伤修复和血管再生等多种生理机能。Gli基因作为Hh信号通路的最终执行者,其异常持续的激活可以启动下游多种与肿瘤相关基因的转录。多项研究结果表明,Gli基因在多种人类恶性肿瘤中异常激活并参与到肿瘤的发生发展及恶性生物学特性之中。研究发现Gli1蛋白在食管鳞癌组织中异常增高表达,与肿瘤的发生形成密切相关,并且可作为评价患者生存预后的独立因素。针对Hh信号通路转录因子Gli的特异性抑制剂GANT61能够诱导恶性间皮瘤、急性髓系白血病、横纹肌肉瘤、胰腺癌等肿瘤细胞的凋亡,目前国内外关于GANT61对人类食管鳞癌作用的研究较为少见,本研究主要探讨Gli抑制剂GANT61对人类食管鳞癌细胞OE21凋亡和侵袭能力的影响,探究食管鳞癌细胞的凋亡机制,寻找低毒高效的食管鳞癌分子靶向药物。方法:采用MTS法检测不同浓度(0、1.171、1.756、2.634、3.951、5.936、8.889、13.333、20、30μmol/L)GANT61处理72h对食管鳞癌细胞OE21的活力抑制作用,绘制细胞活力曲线。计算IC50。采用Western blot方法检测10μmol/L GANT61处理24h对食管鳞癌细胞OE21中Gli1,Fas及cleaved caspase-3蛋白表达的影响。应用流式细胞术检测10μmol/L GANT61处理24h对食管鳞癌细胞OE21细胞凋亡的影响。Transwell侵袭实验检测10μmol/L GANT61处理24h对食管鳞癌细胞OE21侵袭能力的影响。结果:1 MTS实验结果显示,GANT61处理72h对食管鳞癌细胞OE21存在明显细胞毒性作用,显著抑制肿瘤细胞的生长活力,随药物浓度的增加,细胞活力呈现明显下降趋势。IC50=7.975μmol/L。2 Western blot结果显示,与对照组相比,GANT61处理24h能够明显降低食管鳞癌细胞OE21中Gli1蛋白的表达,Fas及cleaved caspase-3凋亡相关蛋白的表达量明显升高。3 流式细胞术检测结果显示,GANT61处理食管鳞癌细胞OE21 24h后可导致细胞凋亡率明显增加,与对照组相比差异有统计学意义(P0.01)。4 Tanswell侵袭实验结果显示,GANT61处理食管鳞癌细胞OE21 24h后,细胞侵袭数目明显减少,与对照组相比差异有统计学意义(P0.01)。结论:Gli1抑制剂GANT61对人类食管鳞癌细胞OE21具有明显的细胞毒性作用,可以显著抑制肿瘤细胞生长活力,且呈明显剂量依赖性,经计算IC50=7.975μmol/L。Western blot结果表明GANT61能够显著抑制Gli1蛋白表达,增加细胞凋亡相关蛋白Fas及cleaved caspase-3的表达量,GANT61可能通过特异性抑制食管鳞癌细胞OE21中Gli1蛋白表达从而减弱其对细胞死亡受体Fas/Fas L凋亡途径的抑制作用,诱导食管鳞癌细胞凋亡。GANT61能够有效抑制食管鳞癌细胞OE21的侵袭转移能力。本研究为食管鳞癌的分子靶向治疗提供理论依据及新的思路。
[Abstract]:Objective: esophageal cancer is one of the common gastrointestinal tumors in China, Chinese patients with esophageal cancer incidence and mortality rate ranks first in the world, the pathological type of esophageal squamous cell carcinoma. The esophageal cancer surgery, radiotherapy chemotherapy effect is poor, the prognosis is not ideal, and to the field of treatment the molecular target of more attention has been paid to.Hedgehog (Hh) signaling a key role in the activation of.Hh signaling pathway in adult tissue stem cell regulation in the process of embryonic development of multicellular organisms in play, tissue repair and vascular regeneration and other physiological functions of Hh signaling pathway.Gli gene as the final executant of its duration the activation of transcription of downstream multiple tumor related genes. Several research results show that the Gli gene in various human malignant tumors and to participate in the abnormal activation of tumor occurrence Development and malignant biological characteristics. The study found that abnormal Gli1 protein expression in esophageal squamous cell carcinoma, and closely related to tumor formation, and can be used as an independent prognostic factor evaluation. A specific inhibitor of GANT61 signaling pathway in Hh transcription factor Gli can induce malignant mesothelioma, acute myeloid leukemia, rhabdomyosarcoma. Apoptosis of pancreatic cancer cells, the current domestic and international research on the effect of GANT61 on human esophageal squamous cell carcinoma is rare, this study aims to investigate the effects of Gli inhibitor GANT61 on human esophageal carcinoma cells OE21 apoptosis and invasion, apoptosis mechanism of esophageal squamous cell carcinoma, squamous cell carcinoma of the esophagus for molecular target of high efficiency and low toxicity to the drug. MTS was used to detect different concentrations (0,1.171,1.756,2.634,3.951,5.936,8.889,13.333,20,30 mol/L) GANT61 72h in esophageal squamous cell carcinoma Inhibition of OE21 cell activity, cell viability curve drawing by Western IC50.. The calculation method for the detection of blot 10 mol/L GANT61 24h of Gli1 OE21 in esophageal squamous cell carcinoma, the expressions of Fas and cleaved caspase-3 protein. Flow cytometry was used to detect 10 mol/L GANT61 treatment effect of 24h on apoptosis of OE21 cells in esophageal squamous cell carcinoma cell line.Transwell invasion assay 10 mol/L GANT61 treatment effect of 24h on the invasion of esophageal squamous cell carcinoma cell line OE21. Results: 1 MTS experimental results show that GANT61 72h has obvious cytotoxic effect on esophageal squamous cell carcinoma OE21 cells, inhibit the growth of tumor cell viability, with the increase in drug concentration, cell viability decreased from.IC50=7.975 mol/L.2 Western blot results showed that compared with the control group, GANT61 24h treatment can significantly reduce the expression of Gli1 protein in esophageal squamous cell carcinoma cell line OE21, Fas The expression of cleaved caspase-3 and apoptosis related protein were significantly increased..3 flow cytometry results showed that GANT61 can lead to cell apoptosis was significantly increased after treatment of esophageal squamous cell carcinoma OE21 24h, compared with the control group had significant difference (P0.01).4 Tanswell invasion experiment results showed that GANT61 treatment of esophageal squamous cell carcinoma cell line OE21 after 24h. The invasion cell number was significantly reduced, compared with the control group the difference was statistically significant (P0.01). Conclusion: the Gli1 inhibitor GANT61 has obvious cytotoxic effect on human esophageal squamous cell carcinoma OE21 cells can significantly inhibit tumor cell growth activity, and a significant dose dependence, the calculation of IC50=7.975 mol/L.Western blot results show that GANT61 can significantly inhibit the expression of Gli1 protein and increasing the expression of apoptosis related protein Fas and cleaved Caspase-3, GANT61 may inhibit the esophageal cell by specific In order to weaken the inhibition of cell death receptor Fas/Fas L apoptosis protein Gli1 expression in cancer cells OE21 induced apoptosis of esophageal squamous cell carcinoma,.GANT61 can effectively inhibit the invasion and metastasis of esophageal squamous cell carcinoma cell line OE21. This study provides a theoretical basis and new ideas for the molecular target of esophageal squamous cell carcinoma to the treatment.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.1

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