【作者】 王林；

【导师】 王雷；

【作者基本信息】 河北医科大学 ， 外科学， 2016， 硕士

【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(P<0.01)。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(P<0.01)。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。更多还原

【Abstract】 Objective: Esophagus cancer is one of the most common gastrointestinal tumor in our country and the morbidity and mortality rate is the highest in the world. The main pathological type of Esophagus is esophageal squamous cell carcinoma. At present, the effect of surgery, radiotherapy and chemotherapy to treat esophagus cancer is not satisfied. The prognosis of patients is very poor. More and more attention has been paid to the treatment of molecular targeted areas. The Hedgehog(Hh) signaling pathway plays a critical role during embryonic development in multicellular organisms. The activation of Hh signaling pathway involves in the regulation of adult stem cells in biological tissue, tissue damage repairment and regeneration of blood vessels and other physiological functions. As a final executor of Hh signaling pathway, continued aberrant activation of Gli causes the transcription of a variety of downstream cancer-related genes. Extensively study show that Gli abnormal activation occurs in a variety of human malignancies and Gli relates to the tumor occurrence and biological characteristics of malignant tumors. It has been discovered that increased abnormal expression of Gli in esophageal squamous biological tissue is closely relevant to the occurrence of tumor, and be considered as a independent factor of evaluation in survival prognosis. GANT6, a specific inhibitor of Hh signaling pathway transcription factor Gli, can induce tumor cells apoptosis of malignant mesothelioma, acute myeloid leukemia, rhabdomyosarcoma, pancreatic cancer and other tumors. Currently, there are very few investigations on GANT61 in human esophageal squamous. Here we mainly investigated the inhibition of esophageal squamous cell growth by GANT61, and the impact of GANT61 on esophageal squamous cell invasion and metastasis, explored the apoptosis mechanism of esophageal squamous cell and seek a low toxicity and efficient targeted drugs to treat esophageal squamous cancer.Methods: Using MTS method to test the viability inhibition of different concentrations of GANT61 treating 72 h of esophageal squamous carcinoma cell OE21(0,1.171,1.756,2.634,3.951,5.936,8.889,13.333,20,30μmol/L), cell viability curve drawing. Get calculation of IC50. Using Western blot method to detect the Gli1, Fas and cleaved caspase 3 protein expression of 10μmol/L GANT61 treating 24 h in esophageal squamous carcinoma cell OE21. Application of FCM to detect 10μmol/L GANT61 role 24 h affect esophageal squamous carcinoma cell OE21 apoptosis. Transwell invasion experiment testing 10μmol/L GANT61 treating 24 h effect on esophageal squamous carcinoma cell OE21 invasion ability.Result:1 The MTS experiment shows that the GANT61 is obvious cytotoxic to esophageal squamous carcinoma cell OE21, inhibit the tumor cell viability, with the increase of drug concentration, the cell viability present obvious downward trend. IC50=7.975μmol/L.2 According to the Western blot result, compared with control group, GANT61 can obviously decrease the Gli1 expression, increase the Fas and cleaved caspase 3 expression.3 The FCM result show that GANT61 treating 24 h in esophageal squamous carcinoma cell OE21 can induce cell apoptosis significantly, compared with control group, the difference was statistically significant(P<0.01).4 Transwell invasion experiment show that GANT61 treating 24 h in esophageal squamous carcinoma cell OE21 the number of cell migration decreased significantly, compared with control group, the difference was statistically significant(P<0.01).Conclusion: Gli1 inhibitor GANT61 is obvious cytotoxic to esophageal squamous carcinoma cell OE21, inhibits the tumor cell viability significantly, and has a dose dependent. IC50=7.975μmol/L. Western blot show that GANT61 can suppress Gli1 protein expression and increase cell apoptosis related protein Fas and cleaved caspase 3 expression, GANT61 may specific inhibit Gli1 protein expression in esophageal squamous carcinoma cell OE21 and decreases the Fas/FasL apoptotic cell death receptor pathway induce the apoptosis of esophageal squamous carcinoma cell. GANT61 can effectively restrain the invasion and metastasis ability of esophageal squamous carcinoma cell OE21. This study provide theoretical basis and new ideas for molecular targeted therapy of esophageal cancer.更多还原