乳酸脱氢酶抑制剂Galloflavin，对子宫内膜癌细胞生物学特性影响及其机制的研究

发布时间：2018-09-19 17:06

【摘要】：目的子宫内膜癌是女性生殖系统常见的恶性肿瘤之一，其发病率逐年升高，严重威胁着女性的生命健康。流行病学统计显示子宫内膜癌高危人群多为肥胖，患有糖尿病，高血压等代谢异常疾病的妇女，而糖代谢与子宫内膜癌的关系也日益得到重视。大多数肿瘤细胞利用糖酵解代谢途径产生的ATP，作为其能量提供的主要方式。靶向针对糖酵解途径抑制肿瘤生长，日益成为治疗肿瘤的新策略。Galloflavin (GF)作为新一代乳酸脱氢酶抑制剂，目前还没有此药物干预子宫内膜癌的基础研究。因此，本研究通过分析GF对人子宫内膜癌细胞株的影响，探讨其作用的可能机制。方法 MTT法观察终浓度为0μmol/L、0.01μmol/L、0.1μmol/L、10μmol/L、50μmol/L、100μmol/L、250μmol/L、500μmol/L GF分别作用于ECC-1、Ishikawa子宫内膜癌细胞株72h后，检测其对细胞增殖的抑制作用。以乳酸脱氢酶试剂盒检测GF干预后乳酸脱氢酶的活性的变化；以乳酸定量试剂盒检测GF干预后乳酸生成量变化；以ATP定量试剂盒检测GF干预后ATP生成量的变化；以2-NBDG检测GF干预后葡萄糖摄取量的变化；以DCFH-DA检测GF干预后细胞内活性氧物质产生量(ROS)的变化；以流式细胞仪、AnnexinV/FITC标记法检测细胞凋亡；以蛋白印迹法分析GF对BCL-2、MCL-1,Cleaved Caspase3蛋白表达的影响。同时行Annexin V-PI染色、流式细胞仪检测草氨酸盐对其凋亡的影响。结果 GF抑制Ishikawa，ECC-1细胞的增殖并且随GF浓度增加，增殖抑制增加，呈剂量依赖性。GF处理细胞后乳酸脱氢酶的活性显著下降，乳酸产生量下降，ATP产生量下降，葡萄糖摄取量下降。活性氧物质产生量增加，Annexin V-PI染色、流式细胞仪检测均表现细胞凋亡比例逐渐增高，证实了GF具有诱导细胞凋亡的作用；GF蛋白印迹检测表明GF可使Cleaved Caspase3表达升高, BCL-2和MCL-1表达下降,GF对人子宫内膜癌细胞株的增殖有显著的抑制作用。结论 GF导致ATP的耗竭，，伴随着细胞内氧化应激产物的增加导致线粒体内凋亡机制的启动，最终导致肿瘤细胞增殖受抑制。靶向针对乳酸脱氢酶治疗子宫内膜癌前景广阔。
[Abstract]:Objective endometrial carcinoma is one of the most common malignant tumors in female reproductive system. Epidemiological statistics show that the high risk population of endometrial cancer is mostly obese, suffering from diabetes, hypertension and other metabolic disorders, and the relationship between glucose metabolism and endometrial carcinoma has been paid more and more attention. Most tumor cells use ATP, produced by glycolysis metabolic pathway as the main way of energy supply. Galloflavin (GF), as a new generation of lactate dehydrogenase inhibitor, has become a new strategy for the treatment of endometrial carcinoma, which is targeted at glycolysis pathway, and there is no basic research on the intervention of Galloflavin (GF) in endometrial carcinoma. In this study, we analyzed the effect of GF on human endometrial cancer cell line and explored its possible mechanism. Methods the inhibitory effects of 0 渭 mol/L,0.01 渭 mol/L,0.1 渭 mol/L,10 渭 mol/L,50 渭 mol/L,100 渭 mol/L,250 渭 mol/L,500 渭 mol/L GF on ECC-1,Ishikawa endometrial carcinoma cell line were observed by MTT assay. Lactate dehydrogenase activity after GF intervention was detected by lactate dehydrogenase kit, lactic acid production after GF intervention was detected by lactic acid quantitative kit, ATP production after GF intervention was detected by ATP quantitative kit. The changes of glucose uptake after GF intervention were detected by 2-NBDG, the changes of reactive oxygen species (ROS) production by GF were detected by DCFH-DA, apoptosis was detected by flow cytometry (FCM) Annexin V / FITC labeling method. The effect of GF on the expression of BCL-2,MCL-1,Cleaved Caspase3 protein was analyzed by Western blot. At the same time, Annexin V-PI staining and flow cytometry were used to detect the effect of oxalate on apoptosis. Results GF inhibited the proliferation of Ishikawa,ECC-1 cells and increased with the increase of GF concentration. In a dose-dependent manner, the activity of lactate dehydrogenase and the production of lactic acid decreased in a dose-dependent manner. Glucose intake decreased. The production of reactive oxygen species increased with Annexin V-PI staining. Flow cytometry showed that the proportion of apoptosis increased gradually, which confirmed that GF could induce apoptosis. GF Western blot analysis showed that GF increased the expression of Cleaved Caspase3 and decreased the expression of BCL-2 and MCL-1. The expression of BCL-2 and MCL-1 decreased significantly and the proliferation of human endometrial cancer cell line was inhibited significantly. Conclusion GF leads to ATP depletion, accompanied by the increase of intracellular oxidative stress products, which leads to the initiation of apoptosis mechanism in mitochondria, and ultimately leads to the inhibition of tumor cell proliferation. Targeted lactate dehydrogenase treatment of endometrial carcinoma has broad prospects.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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