基于组学技术对吖啶酮衍生物8a抗肿瘤机制的研究

发布时间：2018-01-18 15:38

本文关键词：基于组学技术对吖啶酮衍生物8a抗肿瘤机制的研究　出处：《清华大学》2016年博士论文　论文类型：学位论文

【摘要】：实验室合成的吖啶酮类衍生物8a对CCRF-CEM白血病细胞表现出良好的抗肿瘤效果,但对于它的抗肿瘤作用机制仍不清楚。本研究利用组学手段,研究吖啶酮衍生物对CCRF-CEM的作用,从而揭示8a的抗肿瘤分子机制。首先,代谢组学结果表明8a引起23种显著性差异的代谢物涉及5条代谢途径。其中在谷胱甘肽代谢通路中,8a引起还原型谷胱甘肽的降低和L-半胱氨酸-甘氨酸和谷氨酸的显著升高;在甘油磷脂代谢通路中,细胞膜主要成分甘油磷脂明显升高而溶血卵磷脂明显降低。此外,还发现8a引起ROS和脂质过氧化的副产物MDA水平均明显升高,并伴随有线粒体膜电位的降低,细胞色素C的释放和Caspase-3的激活,最终导致了细胞的凋亡。其次,蛋白质组学结果表明8a影响了很多的细胞通路:包括染色体组装,能量代谢,DNA损伤,氧化应激和凋亡途径。通过PKM-2验证组学研究中的蛋白表达变化。此外,8a不仅抑制了HEX和PFK-1的蛋白表达,也显著降低了乳酸的生成量。这些都证明糖酵解过程被8a显著抑制。升高的XRCC6和降低的组蛋白也表明DNA损伤可能发生在8a作用的细胞中,而这些假设通过升高的γ-H2AX被证明。分子对接表明8a和DNA的相互作用方式包括3种氢键和4种π π相互作用,从而引起了DNA的损伤。第三,全细胞的糖组学分析一共鉴定出135种不同结构的N糖和83种不同组成的N糖,其中有7种高甘露糖型的N糖在8a的刺激下呈现明显降低的趋势。细胞膜部分的糖组学分析一共鉴定出360种不同结构的N糖和203种不同组成的N糖,其中一共有42种N糖在8a刺激前后呈现显著性差异变化,这包括12种高甘露型,4种非修饰的复合/杂合型,9种岩藻糖型和9种唾液酸型N糖。相比起全细胞裂解液来说,细胞膜部分能更敏感的监控吖啶酮药物刺激下的糖基化变化。而这所有的糖基化变化被表明是和8a抑制了负责催化糖链转移的酶DDOST有关。综上所述,8a可能是通过下面多种机制来引发肿瘤细胞的死亡:氧化应激调节的凋亡,能量途径的紊乱,DNA的损伤等等。综合以上多种组学手段的研究,不仅对8a的抗肿瘤作用机理提供了新颖的见解,也有助于加快8a结构的进一步优化。
[Abstract]:The synthetic acridine ketone derivatives 8 a showed good antitumor effect on CCRF-CEM leukemia cells, but the mechanism of its antitumor effect was still unclear. To study the effect of acridine ketone derivatives on CCRF-CEM, so as to reveal the anti-tumor molecular mechanism of 8a. The results of metabonomics showed that the 23 metabolites which caused significant difference in 8 years were involved in 5 metabolic pathways, among which glutathione pathway was involved. 8a induced reduced glutathione and increased L-cysteine-glycine and glutamate. In glycerol phospholipid metabolism pathway, the main component of cell membrane glycerol phospholipid increased significantly, and lysophosphatidylcholine decreased significantly. It was also found that the level of MDA, a by-product of ROS and lipid peroxidation, was significantly increased after 8 years, accompanied by the decrease of mitochondrial membrane potential, the release of cytochrome C and the activation of Caspase-3. Secondly, proteomics results show that 8a affects many cell pathways, including chromosome assembly, energy metabolism and DNA damage. Oxidative stress and apoptotic pathway. The protein expression in PKM-2 was verified by PKM-2. In addition, the protein expression of HEX and PFK-1 was not only inhibited by T8a. These results showed that the glycolysis process was significantly inhibited by 8a. The increased XRCC6 and reduced histone also suggested that DNA damage might occur in the cells treated for 8 years. These hypotheses are proved by elevated 纬 -H2AX. Molecular docking indicates that the interaction between 8a and DNA consists of three hydrogen bonds and four 蟺蟺 interactions. This caused the damage of DNA. Thirdly, a total of 135 kinds of N-sugar with different structure and 83 kinds of N-sugar with different composition were identified by the whole cell glycometry analysis. Seven kinds of high mannose type N sugar showed a decreasing trend after 8 years of stimulation. A total of 360 different structure N sugars and 203 different composition N sugars were identified by the glycometological analysis of the cell membrane. A total of 42 N sugars showed significant differences before and after 8 years of stimulation, including 12 high mannose types and 4 non-modified composite / heterozygous types. Nine kinds of fucose and nine kinds of sialic acid N sugar. Compared with the whole cell lysate. The membrane part can more sensitively monitor the glycosylation changes stimulated by acridine ketones. All these glycosylation changes have been shown to be related to the inhibition of the enzyme DDOST, which catalyzes the sugar chain transfer. 8a may induce the death of tumor cells through the following mechanisms: apoptosis regulated by oxidative stress, DNA damage caused by energy pathway disorder, etc. It not only provides new insights into the anti-tumor mechanism of 8a, but also accelerates the further optimization of 8a structure.
【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96

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