葡萄糖浓度对子宫内膜癌细胞生长的影响及其机制研究
发布时间：2018-08-29 18:38
【摘要】：目的:子宫内膜癌(EC)是最常见的女性恶性肿瘤之一,其发病率和病死率逐年升高。肥胖是促使Ⅰ型EC进展的高危因素,与EC的高病死率密切相关。多项研究结果显示,细胞代谢异常和胰腺癌、结直肠癌和乳腺癌等多种肿瘤的发病密切相关,与EC的关系也日益获得关注。糖代谢异常是超重、肥胖和糖尿病的病理特征之一,也是EC发病的重要危险因素。但糖代谢异常促进EC发生的作用机制尚未明了。因此,在本研究中,我们应用已建立的EC细胞系(Ishikawa、HEC-1-B),观察在体外不同葡萄糖浓度培养条件下EC细胞增殖情况及其机制探讨。方法:通过体外培养EC细胞株Ishikawa和HEC-1-B,用含不同糖浓度(无糖组:0mmol/L、低糖组:2.5mmol/L、正常糖浓度组:5.5mmol/L、高糖组:25.5mmol/L)的DMEM完全培养液处理细胞,使用CCK-8法来评价不同糖浓度对EC细胞增殖的影响;使用PI染色法和Annexin V/PI法检测不同糖浓度对EC细胞周期和凋亡的影响;使用DCFH-DA荧光探针来测定不同糖浓度引起的EC细胞活性氧(ROS)的改变;使用ATP发光检测试剂盒来检测不同糖浓度对EC细胞产生ATP的影响;通过Western蛋白印迹实验检测上述EC细胞在不同糖浓度干预下,AMPK和AKT/mTOR/S6信号通路相关蛋白的表达变化,以明确在不同的代谢环境下细胞代谢的信号转导通路。结果:在不同糖浓度条件下培养EC细胞系Ishikawa和HEC-1-B,发现2株EC细胞的增殖均依赖于培养基中的葡萄糖浓度,且呈剂量依赖模式;低糖浓度通过G1期阻滞抑制Ishikawa和HEC-1-B细胞增殖,高糖浓度抑制细胞凋亡;低糖浓度改变糖酵解活性,引起Ishikawa和HEC-1-B细胞ATP生成减少,ROS产生增加;Western蛋白印迹实验结果显示,在Ishikawa和HEC-1-B细胞中,低糖增加p-AMPK的表达,降低p-AKT及p-S6的表达,证实糖代谢异常通过调节AMPK/mTOR和Akt/mTOR/S6信号通路促进EC细胞增殖。结论:发现EC细胞的增殖以剂量依赖性方式依赖于培养基中的葡萄糖浓度,糖代谢异常通过调节AMPK/mTOR和Akt/mTOR/S6信号通路促进EC细胞增殖,靶向糖代谢异常及其相关信号通路可能为肥胖型EC患者个体化治疗提供新思路。
[Abstract]:Objective: (EC) is one of the most common malignant tumors in women. Obesity is a high risk factor for the progression of type I EC and is closely related to the high mortality of EC. Many studies have shown that abnormal cell metabolism is closely related to pancreatic cancer, colorectal cancer, breast cancer and other tumors, and the relationship with EC has been paid more and more attention. Abnormal glucose metabolism is one of the pathological features of overweight, obesity and diabetes, and is also an important risk factor of EC. However, the mechanism of abnormal glucose metabolism promoting the occurrence of EC is not clear. Therefore, in this study, we used the established EC cell line (Ishikawa,HEC-1-B) to observe the proliferation of EC cells under different glucose concentrations in vitro and its mechanism. Methods: EC cell lines Ishikawa and HEC-1-B, were treated with DMEM complete culture medium containing different sugar concentrations (no sugar group: 0 mmol / L, low sugar group: 2.5 mmol / L, normal glucose concentration group: 5. 5 mmol / L, high glucose group: 25. 5 mmol / L). The effect of different glucose concentrations on the proliferation of EC cells was evaluated by CCK-8 method. PI staining and Annexin V/PI assay were used to detect the effect of different glucose concentrations on the cell cycle and apoptosis of EC cells, and DCFH-DA fluorescence probe was used to detect the changes of reactive oxygen (ROS) (Ros) in EC cells induced by different glucose concentrations. The effects of different glucose concentrations on the production of ATP in EC cells were detected by ATP luminescence assay kit, and the expression of AMPK and AKT/mTOR/S6 signaling pathway related proteins in EC cells were detected by Western Western blot. In order to clarify the signal transduction pathway of cell metabolism in different metabolic environment. Results: the proliferation of EC cell lines Ishikawa and HEC-1-B, in different glucose concentration was found to be dependent on glucose concentration in culture medium, and low glucose concentration inhibited the proliferation of Ishikawa and HEC-1-B cells through G1 phase arrest. High glucose concentration inhibited cell apoptosis, low glucose concentration changed glycolysis activity, reduced ATP production in Ishikawa and HEC-1-B cells, and increased Ros production. Western blot analysis showed that low glucose increased p-AMPK expression in Ishikawa and HEC-1-B cells. Decreasing the expression of p-AKT and p-S6 showed that abnormal glucose metabolism promoted the proliferation of EC cells by regulating the signal pathway of AMPK/mTOR and Akt/mTOR/S6. Conclusion: the proliferation of EC cells depends on glucose concentration in a dose-dependent manner. Abnormal glucose metabolism promotes the proliferation of EC cells by regulating AMPK/mTOR and Akt/mTOR/S6 signaling pathways. The abnormal glucose metabolism and its related signaling pathway may provide a new idea for individual treatment of obese EC patients.
【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】：R737.33
本文编号：2212065
[Abstract]:Objective: (EC) is one of the most common malignant tumors in women. Obesity is a high risk factor for the progression of type I EC and is closely related to the high mortality of EC. Many studies have shown that abnormal cell metabolism is closely related to pancreatic cancer, colorectal cancer, breast cancer and other tumors, and the relationship with EC has been paid more and more attention. Abnormal glucose metabolism is one of the pathological features of overweight, obesity and diabetes, and is also an important risk factor of EC. However, the mechanism of abnormal glucose metabolism promoting the occurrence of EC is not clear. Therefore, in this study, we used the established EC cell line (Ishikawa,HEC-1-B) to observe the proliferation of EC cells under different glucose concentrations in vitro and its mechanism. Methods: EC cell lines Ishikawa and HEC-1-B, were treated with DMEM complete culture medium containing different sugar concentrations (no sugar group: 0 mmol / L, low sugar group: 2.5 mmol / L, normal glucose concentration group: 5. 5 mmol / L, high glucose group: 25. 5 mmol / L). The effect of different glucose concentrations on the proliferation of EC cells was evaluated by CCK-8 method. PI staining and Annexin V/PI assay were used to detect the effect of different glucose concentrations on the cell cycle and apoptosis of EC cells, and DCFH-DA fluorescence probe was used to detect the changes of reactive oxygen (ROS) (Ros) in EC cells induced by different glucose concentrations. The effects of different glucose concentrations on the production of ATP in EC cells were detected by ATP luminescence assay kit, and the expression of AMPK and AKT/mTOR/S6 signaling pathway related proteins in EC cells were detected by Western Western blot. In order to clarify the signal transduction pathway of cell metabolism in different metabolic environment. Results: the proliferation of EC cell lines Ishikawa and HEC-1-B, in different glucose concentration was found to be dependent on glucose concentration in culture medium, and low glucose concentration inhibited the proliferation of Ishikawa and HEC-1-B cells through G1 phase arrest. High glucose concentration inhibited cell apoptosis, low glucose concentration changed glycolysis activity, reduced ATP production in Ishikawa and HEC-1-B cells, and increased Ros production. Western blot analysis showed that low glucose increased p-AMPK expression in Ishikawa and HEC-1-B cells. Decreasing the expression of p-AKT and p-S6 showed that abnormal glucose metabolism promoted the proliferation of EC cells by regulating the signal pathway of AMPK/mTOR and Akt/mTOR/S6. Conclusion: the proliferation of EC cells depends on glucose concentration in a dose-dependent manner. Abnormal glucose metabolism promotes the proliferation of EC cells by regulating AMPK/mTOR and Akt/mTOR/S6 signaling pathways. The abnormal glucose metabolism and its related signaling pathway may provide a new idea for individual treatment of obese EC patients.
【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】：R737.33
【参考文献】
相关期刊论文 前2条
1 JI Cheng Ye;CHEN Tian Jiao;;Empirical Changes in the Prevalence of Overweight and Obesity among Chinese Students from 1985 to 2010 and Corresponding Preventive Strategies[J];Biomedical and Environmental Sciences;2013年01期
2 杨华;谭先杰;郎景和;;代谢综合征与子宫内膜癌相关性研究进展[J];中国实用妇科与产科杂志;2012年11期
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