【作者】 徐浩；

【导师】 王伟林；

【作者基本信息】 浙江大学 ， 临床医学（专业学位）， 2019， 硕士

【摘要】 研究背景:胰腺癌是最常见的消化道肿瘤之一,其发病率逐年增加。由于早期缺乏典型的临床症状,大多数患者就诊时往往出现肿瘤周围浸润和远处器官的转移,预后极差。据美国癌症协会预测,胰腺癌将成为第三大肿瘤相关致死病因,在世界范围内每年造成227000人死亡。尽管基础研究的深入和医学标准的进步,基于肿瘤手术切除的综合治疗策略取得了良好的效果,但胰腺癌病患的5年生存率仍然小于8%。面对严峻的形势,创新有效的治疗策略迫在眉睫。EZH2 全称 Enhancer of Zeste Homolog 2,是多梳蛋白抑制复合物 2(polycomb repressive complex 2,PRC2)的催化亚基,能催化组蛋白H3第27赖氨酸位点的三甲基化,从而在转录水平引起靶基因的沉默。大量文献报道,EZH2在多种肿瘤的进展中发挥着重要的作用,如乳腺癌、前列腺癌、子宫内膜癌、黑色素瘤和血液系统的恶性肿瘤等。GSK343是一种选择性的EZH2抑制剂,能够通过竞争抑制甲基供体S-腺苷-L-甲硫氨酸(S-Adenosyl-L-Methionine,SAM)来发挥作用。而EZH2在癌症中高表达,与肿瘤的预后与转移有着密切的关系,抑制EZH2被认为是一种新颖的潜在的治疗癌症的方向。在本研究中,我们选用了两株胰腺癌细胞株AsPC-1和PANC-1,旨在探究EZH2抑制剂GSK343在胰腺癌中的作用及其机制,为胰腺肿瘤的治疗提供新思路和方法。研究方法:1、GSK343在不同胰腺癌细胞系中IC50的确定不同浓度的GSK343处理胰腺癌细胞,运用MTT法检测GSK343在胰腺癌细胞系AsPC-1和PANC-1中的IC50浓度,并绘制细胞活力-药物浓度曲线。2、GSK343对于胰腺癌细胞生物学行为的影响1)选择合适的浓度,分别运用CCK-8和EdU实验检测GSK343对胰腺癌细胞增殖能力影响。2)选择适宜的浓度,采用流式细胞术分析GSK343对胰腺癌细胞凋亡的影响。3)选择恰当的浓度,使用流式细胞术分析GSK343对胰腺癌细胞周期的影响。3、GSK343诱导胰腺癌细胞自噬及其机制探索1)运用免疫荧光技术,在荧光显微镜下观察LC3B-Ⅱ的浓聚,检测GSK343对于胰腺癌细胞自噬的影响。2)选择合适的浓度,Western-blotting方法检测ATG5和LC3B的表达,从而检测GSK343诱导胰腺癌细胞自噬的能力。3)加入合适的GSK343后,继续采用Western-blotting方法检测AKT、p-AKT(ser473)、mTOR及p-mTOR(ser2448)的表达,探索GSK343诱导胰腺癌细胞自噬的可能机制。实验结果:1、GSK343在不同胰腺癌细胞系中IC50值MTT方法显示,GSK343在胰腺癌细胞系 AsPC-1 中的IC50值为12.71±0.41μmol/L,而在胰腺癌细胞系 PANC-1 中的 IC50 值为 12.04± 1.10μmol/L,两者之间没有明显统计学差异。2、GSK343对于胰腺癌细胞生物学行为的影响CCK-8及EdU实验显示,加入GSK343后两种胰腺癌细胞系增殖能力显著降低;流式细胞术分析发现,GSK343能够增加胰腺癌细胞的凋亡率,阻滞细胞周期于GO/G1期。3、GSK343诱导胰腺癌细胞自噬及其机制探索1)免疫荧光技术发现,加入合适浓度的GSK343后,LC3B-Ⅱ显著浓聚,而Western-blotting方法检测显示,ATG5的表达未发生变化,LC3B-Ⅰ的表达显著下降,而LC3B-Ⅱ的表达显著上升,LC3B-Ⅱ/LC3B-Ⅰ 比值增大。2)适宜的浓度处理胰腺癌细胞后,Western-blotting方法显示AKT及mTOR的表达未发生明显变化,而p-AKT(ser473)及p-mTOR(ser2448)的表达显著降低。实验结论:1、GSK343作用于胰腺癌细胞系AsPC-1和PANC-1中的IC50值相似,对胰腺癌细胞有普遍的杀伤作用,在胰腺癌的临床治疗中有一定的应用前景。2、GSK343能够有效抑制胰腺癌细胞的增殖,诱导胰腺癌细胞的凋亡,并阻滞细胞周期于G0/G1期。3、GSK343能够诱导胰腺癌细胞自噬并下调AKT/mTOR信号通路,这可能是GSK343发挥抑癌作用的重要机制之一。更多还原

【Abstract】 ObjectivesBeing one of the most common digestive tract malignant tumor,the incidence of pancreatic cancer is ascending year after year.Due to the lack of typical clinical symptoms in early-stage,most patients often go to hospital after the tumor invades surrounding tissues or metastases to distant organs,and the prognosis is extremely poor.According to predictions by the American Cancer Society,pancreatic cancer will be the third leading cause of cancer death in the USA and lead to an estimated 227 000 deaths per year worldwide.With the deeper recognition of tumors and advance of medical technology,comprehensive therapeutic strategies based on the operations have acquired better effect for cancer treatment.Nevertheless,patients’ five-year survival rate is still only 8%.Faced with such grim situation,effective and innovative treatment strategies are extremely urgentEnhancer of zeste homolog 2(EZH2),which is the enzymatically active subunit of polycomb repressive complex 2(PRC2),can catalyze trimethylation of lysine 27 on histone 3(H3K27),leading to transcriptional silencing of target genes.A large number of studies have reported that EZH2 plays a key role in the progression of various cancers,such as prostate cancer,breast cancer,endometrial cancer,melanoma,and hematological malignancies.GSK343 acts as a SAM-competitive inhibitor to exert an enzymatically repressive function on EZH2.The high expression of EZH2 in cancer is closely related to the prognosis and metastasis of cancer.Inhibition of EZH2 is regarded as a novel and potential therapeutic target for carcinomas.In this study,we selected two pancreatic cancer cell lines AsPC-1 and PANC-1 to explore the function and mechanism of EZH2 inhibitor GSK343,and may provide a new strategy and method for the treatment of pancreatic tumors.Methods1.Determination of IC50 in GSK343 in different pancreatic cancer cell linesAfter treated with different concentrations of GSK343,the IC50 concentration of GSK343 in pancreatic cancer cell lines AsPC-1 and PANC-1 was detected by MTT assay,and then draw the cell viability-drug concentration curve.2.The effects of GSK343 on cell biological behavior of pancreatic cancer cells1)The effect of GSK343 on the proliferation of pancreatic cancer cells was determined by CCK-8 and EdU assay.2)Select appropriate concentration and analyze the effect of GSK343 on apoptosis of pancreatic cancer cells by flow cytometry.3)Select the suitable concentration and use flow cytometry to analyze the effect of3.The effects of GSK343 on autophagyGSK343 on pancreatic cancer cell cycle.in pancreatic cancer cells and its mechanism1)Using immunofluorescence technique,the puncta of LC3B-Ⅱ was observed under a fluorescence microscope to examine the effect of GSK343 on autophagy in pancreatic cancer cells.2)Select the appropriate concentration and detect the ATG5 and LC3B by Western-blotting assay to confirm the ability of GSK343 on autophagy in pancreatic cancer cells.3)After adding appropriate concentration of GSK343,Western blotting was used to detect AKT,p-AKT(ser473),mTOR and p-mTOR(ser2448),and explore the possible mechanism of GSK343-induced autophagy in pancreatic cancer cells.Results1.IC50 values of GSK343 in different pancreatic cancer cell linesThe MTT method showed that the IC50 value of GSK343 in the pancreatic cancer cell line AsPC-1 was 12.71±0.41μmol/l,while the IC50 value in the pancreatic cancer cell line PANC-1 was 12.04±1.10μmol/l.There were no significant statistical differences.2.The effect of GSK343 on the biological behavior of pancreatic cancer cellsCCK-8 and EdU assay showed that the proliferation of the two pancreatic cancer cell lines was significantly decreased after the addition of GSK343.Flow cytometry analysis showed that GSK343 could increase the apoptosis rate of pancreatic cancer cells and arrest the cell cycle in G0/G1 phase.3.GSK343 induces autophagy in pancreatic cancer cells and its mechanism1)Immunofluorescence technique showed that the puncta of LC3B-Ⅱ significantly increased after adding appropriate concentration of GSK343,meanwhile Western-blotting showed that the expression of ATG5 did not change,LC3B-Ⅰ decreased,and LC3B-Ⅱ increased,and the ratio of LC3B-Ⅱ/LC3B-Ⅰ augmented.2)After treatment of pancreatic cancer cells at a suitable concentration,Western-blotting showed that the expression of AKT and mTOR unchanged,while the expression of p-AKT(ser473)and p-mTOR(ser2448)was significantly decreased.Conclusions1)The IC50 values of GSK343 in pancreatic cancer cell lines AsPC-1 and PANC-1 are no statistically significant difference,which have a general killing effect on pancreatic cancer cells,and have certain application prospects in the clinical treatment of pancreatic cancer.2)GSK343 can effectively inhibit the proliferation of pancreatic cancer cells,induce apoptosis of pancreatic cancer cells,and block the cell cycle in G0/G1 phase.3)GSK343 can induce autophagy in pancreatic cancer cells and down-regulate AKT/mTOR signaling pathway,which may be one of the important mechanisms of GSK343 to play a role in tumor suppression.更多还原