LSD1抑制剂SDZ17诱导胃癌细胞的代谢重编程机制的研究

发布时间：2018-04-09 14:41

本文选题：LSD1抑制剂　切入点：代谢组学　出处：《郑州大学》2017年硕士论文

【摘要】：我国胃癌的发病率居高不下,患者多处于局部进展期和晚期且生存率较低,早期胃癌的表现与胃炎类似,其早期诊断和精准治疗都比较困难。胃癌的发生发展是一个多因素参与的复杂生物学过程,它发生、发展的病理变化往往造成机体基础代谢产生相应改变,进而引起小分子代谢物种类或浓度随着时间的变化而发生相应的变化,最终造成与正常个体之间代谢轮廓的差异。赖氨酸特异性去甲基化酶1(LSD1)是第一个被发现的组蛋白去甲基化酶,是细胞内控制很多生命过程的开关,与胃癌的发生、侵袭密切相关。胃癌的发生和转移往往伴随着LSD1的异常表达,基于此,本论文选用LSD1异常表达的胃癌细胞株MGC-803和正常表达的胃粘膜上皮细胞株GES-01作为研究对象,采用SDZ17对其进行干扰,运用动态代谢组学技术,对胃癌细胞的代谢通路进行分析;拟从分子水平上揭示LSD1在胃癌细胞代谢过程中的重要调控作用;通过统计学分析得到胃癌细胞的分子标志物,为胃癌的早期诊断、靶向药物精准治疗提供基础理论依据。一、论文实验内容主要完成以下部分:(1)采用MTT法测定LSD1抑制剂SDZ17对LSD1高表达的胃癌细胞MGC-803和胃粘膜上皮细胞GES-01增殖抑制作用的研究;(2)基于UPLC-QEMS测定细胞代谢物的方法学研究;(3)代谢组学技术研究SDZ17对LSD1高表达的胃癌细胞MGC-803和胃粘膜上皮细胞GES-01的代谢重编程作用。二、结果:(1)采用MTT法测定了SDZ17对处于对数期生长期的胃癌细胞MGC-803和胃粘膜上皮细胞GES-01的生长抑制率,半数抑制浓度分别为1.0μM和50.0μM;SDZ17对MGC-803的增殖抑制作用较GES-01强,且对两种细胞的抑制作用具有时间和浓度依赖性。(2)根据对细胞代谢物的提取率和稳定性优选了甲醇-水-氯仿(12:3:5,v/v/v)作为萃取剂系统;根据色谱柱压力,多针连续进样的稳定性以及QC样品的重现性优化了色谱条件,通道A(水)和通道B(乙腈)的梯度洗脱体系满足要求。方法学考察结果表明该方法操作简单,重现性好,通量高,能够高效稳定地萃取和保留细胞样品中的小分子代谢物,符合方法学的要求。(3)SDZ17对胃癌细胞MGC-803的代谢调控作用研究结果显示:SDZ17对LSD1高表达的胃癌细胞MGC-803的代谢通路具有较强的影响,可引起包括氨酰tRNA生物合成、精氨酸和脯氨酸代谢途径、氮平衡、缬氨酸,异亮氨酸和亮氨酸代谢在内的代谢通路的变化;SDZ17诱导胃癌细胞的发生代谢重编程,且具有时间和浓度依赖性。(4)SDZ17对正常胃粘膜上皮细胞GES-01的代谢调控作用研究的结果显示:SDZ17对正常胃粘膜上皮细胞GES-01的代谢通路的影响相对于胃癌细胞MGC-803的影响较小,且随着作用时间的延长,影响作用有趋于减弱的态势。三、结论:(1)LSD1抑制剂对LSD1高表达的胃癌细胞MGC-803的增殖具有抑制作用。(2)LSD1可能是诱导胃癌细胞MGC-803代谢重编程的重要因素之一。
[Abstract]:The incidence of gastric cancer in China is high, most of the patients are in local advanced and advanced stage, and the survival rate is low. The early gastric cancer is similar to gastritis, and its early diagnosis and accurate treatment are difficult.The occurrence and development of gastric cancer is a complicated biological process in which many factors are involved. The pathological changes in the occurrence and development of gastric cancer often result in corresponding changes in basic metabolism of the body.Then the type or concentration of metabolites of small molecules changes with time, resulting in the difference of metabolic profile between individuals and normal individuals.Lysine-specific demethylase 1 (LSD1) is the first histone demethylase found, which is a switch that controls many life processes in cells and is closely related to the occurrence and invasion of gastric cancer.The occurrence and metastasis of gastric cancer are often accompanied by abnormal expression of LSD1. In this paper, the abnormal expression of LSD1 in gastric cancer cell line MGC-803 and normal expression of gastric mucosal epithelial cell line GES-01 were selected as the research object, and SDZ17 was used to interfere with the expression of MGC-803.The metabolic pathway of gastric cancer cells was analyzed by using dynamic metabolomics technology; the important regulatory role of LSD1 in the metabolic process of gastric cancer cells was revealed at the molecular level; the molecular markers of gastric cancer cells were obtained by statistical analysis.To provide basic theoretical basis for early diagnosis of gastric cancer and targeted drug therapy.First,The main contents of this paper are as follows: (1) MTT assay was used to determine the inhibitory effect of LSD1 inhibitor SDZ17 on the proliferation of gastric cancer cells MGC-803 and GES-01, which is highly expressed by LSD1.To study the metabolic reprogramming effect of SDZ17 on MGC-803 and GES-01 of gastric cancer cells with high expression of LSD1.Results: MTT method was used to determine the growth inhibition rate of SDZ17 on MGC-803 and GES-01 of gastric cancer cells in logarithmic phase. The inhibitory concentration of 50 渭 M and 50 渭 M SDZ17 was 1.0 渭 M and 50.0 渭 M, respectively, and the inhibitory effect of SDZ17 on MGC-803 proliferation was stronger than that on GES-01.And the inhibitory effects on the two kinds of cells were time-and concentration-dependent.) according to the extraction rate and stability of the metabolites of the cells, methanol-water-chloroform (12: 3: 5: 5 v / v / v) was selected as the extractant system, according to the chromatographic column pressure,The stability of multi-needle continuous injection and the reproducibility of QC sample were optimized. The gradient elution system of channel A (water) and channel B (acetonitrile) satisfied the requirements.The results showed that the method was simple, reproducible and high-throughput, and could extract and retain the metabolites of small molecules in cell samples efficiently and stably.The results of the study on the metabolic regulation of MGC-803 in gastric cancer cells showed that: 1. SDZ17 had a strong effect on the metabolic pathway of MGC-803 in gastric cancer cells with high LSD1 expression, which could induce the biosynthesis of tRNA, including the biosynthesis of aminoacyl tRNA.Changes in the metabolic pathways including arginine and proline metabolism, nitrogen balance, valine, isoleucine and leucine metabolism. SDZ17 induced metabolic reprogramming in gastric cancer cells.The results showed that the effect of GES-01 on the metabolic pathway of GES-01 in normal gastric epithelial cells was less than that of MGC-803 in normal gastric epithelial cells.And with the extension of action time, the effect tends to weaken.Conclusion: the inhibition of the proliferation of gastric cancer cell line MGC-803 with high expression of LSD1 may be one of the important factors that induce the metabolic reprogramming of gastric cancer cell line MGC-803.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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