糖酵解及抑制剂影响卵巢癌生长增殖机制的体外实验研究

发布时间：2018-04-16 06:08

本文选题：卵巢癌 + 葡萄糖　；参考：《济南大学》2017年硕士论文

【摘要】：目的近年来流行病学证据表明,肥胖可能通过上调体内胰岛素及胰岛素样生长因子水平以及多种脂肪因子相互作用,改变卵巢癌的发病机制。癌细胞的细胞生长与分裂需要通过其自身新陈代谢提供中间物质,并且维持其自身于较低水平的氧化(还原)稳态。本研究旨在通过探讨糖酵解途径与谷氨酰胺分解途径之间可能存在的潜在关系,从而分别验证葡萄糖与谷氨酰胺对卵巢癌细胞生长的影响。针对SKOV3,IGROV-1与Hey三种卵巢癌细胞系,给予不同刺激后,通过分析其细胞毒性、细胞周期、细胞凋亡与细胞内ATP的生物合成的改变,探讨葡萄糖、谷氨酰胺与相应抑制剂对卵巢癌细胞生长的影响。方法应用MTT法、细胞周期分析、膜联蛋白V测定、裂解胱天蛋白酶3的测定实验、Annexin-PI法、流式细胞仪,检测在不同葡萄糖浓度、谷氨酰胺以及丙酮酸培养条件下,以及经2-脱氧葡萄糖(2-DG)、3-溴丙酮酸(3-BP)与AOA行不同干预后,卵巢癌细胞在增殖、凋亡、细胞周期、细胞活性等方面的改变。通过应用ATP生物发光检测试剂盒观察不同糖浓度刺激后,卵巢癌细胞内ATP产量的变化。运用乳酸定量试剂盒以检测4种葡萄糖浓度对卵巢癌细胞中乳酸盐生成的影响。通过应用免疫蛋白印迹法检测在不同的培养条件对P-MAPK、P-S6、CDK4、cyclinD1、P21、Mcl-1、Bcl-2等相关蛋白表达的影响。结果葡萄糖与谷氨酰胺为卵巢癌细胞提供主要的能量来源。葡萄糖促进三种卵巢癌细胞的细胞增殖、增加其胞内ATP水平与乳酸盐产量。葡萄糖、谷氨酰胺以及丙酮酸三者联合能够最大程度刺激卵巢癌细胞增殖。低糖或无糖条件诱导卵巢癌细胞的凋亡,使膜联蛋白V表达增加,半胱天冬酶-3活性增加。葡萄糖亦诱导卵巢癌细胞周期阻滞。2-DG,3-BP与AOA抑制卵巢癌细胞增殖。2-DG诱导的卵巢癌细胞阻滞于G1期。AOA增加卵巢癌细胞对2-DG的敏感性,二者协同抑制卵巢癌的细胞增殖。结论葡萄糖是卵巢癌细胞最重要的能量来源。糖酵解抑制剂联合AOA可能对卵巢癌的治疗具有新的应用价值。
[Abstract]:Objective recent epidemiological evidence suggests that obesity may change the pathogenesis of ovarian cancer by upregulating the levels of insulin, insulin-like growth factor and multiple fatty factors.The growth and division of cancer cells need to provide intermediates through their own metabolism and maintain a low level of redox homeostasis.The purpose of this study was to investigate the potential relationship between glycolysis pathway and glutamine decomposition pathway and to verify the effects of glucose and glutamine on the growth of ovarian cancer cells.Three ovarian cancer cell lines SKOV3, IGROV-1 and Hey were treated with different stimuli. The changes of cytotoxicity, cell cycle, apoptosis and intracellular ATP biosynthesis were analyzed.Effects of glutamine and its inhibitors on the growth of ovarian cancer cells.Methods MTT assay, cell cycle analysis, membrane protein V assay, cystatin 3 assay, Annexin-PI assay and flow cytometry were used to detect the concentration of glucose, glutamine and pyruvate.After different intervention of 2-DGG 3-BPwith AOA, the changes of proliferation, apoptosis, cell cycle and cell activity of ovarian cancer cells were observed.ATP bioluminescence assay kit was used to observe the changes of ATP production in ovarian cancer cells stimulated by different glucose concentrations.The effects of four glucose concentrations on lactate production in ovarian cancer cells were detected by using lactic acid quantitative kit.The effects of different culture conditions on the expression of cyclin D1OP21mcl-1Bcl 2 and other related proteins were detected by Western blot.Results glucose and glutamine provided the main energy source for ovarian cancer cells.Glucose promoted the proliferation of three kinds of ovarian cancer cells and increased the intracellular ATP level and lactate production.The combination of glucose, glutamine and pyruvate can stimulate the proliferation of ovarian cancer cells to the greatest extent.The apoptosis of ovarian cancer cells was induced by low glucose or no glucose condition, the expression of annexin V was increased, and the activity of cysteinase-3 was increased.Glucose also induced ovarian cancer cell cycle arrest. 2-DGG 3-BP and AOA inhibited the proliferation of ovarian cancer cells. 2 DG induced inhibition of ovarian cancer cell proliferation at G1 phase. AOA increased the sensitivity of ovarian cancer cells to 2-DG, and both inhibited the proliferation of ovarian cancer cells.Conclusion glucose is the most important energy source for ovarian cancer cells.Glycolytic inhibitors combined with AOA may be of new value in the treatment of ovarian cancer.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31

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