本文關(guān)鍵詞：基于組學(xué)技術(shù)對吖啶酮衍生物8a抗腫瘤機制的研究　出處：《清華大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 吖啶酮衍生物 代謝組學(xué) 蛋白質(zhì)組學(xué) 糖組學(xué) 機制研究

【摘要】：實驗室合成的吖啶酮類衍生物8a對CCRF-CEM白血病細胞表現(xiàn)出良好的抗腫瘤效果,但對于它的抗腫瘤作用機制仍不清楚。本研究利用組學(xué)手段,研究吖啶酮衍生物對CCRF-CEM的作用,從而揭示8a的抗腫瘤分子機制。首先,代謝組學(xué)結(jié)果表明8a引起23種顯著性差異的代謝物涉及5條代謝途徑。其中在谷胱甘肽代謝通路中,8a引起還原型谷胱甘肽的降低和L-半胱氨酸-甘氨酸和谷氨酸的顯著升高;在甘油磷脂代謝通路中,細胞膜主要成分甘油磷脂明顯升高而溶血卵磷脂明顯降低。此外,還發(fā)現(xiàn)8a引起ROS和脂質(zhì)過氧化的副產(chǎn)物MDA水平均明顯升高,并伴隨有線粒體膜電位的降低,細胞色素C的釋放和Caspase-3的激活,最終導(dǎo)致了細胞的凋亡。其次,蛋白質(zhì)組學(xué)結(jié)果表明8a影響了很多的細胞通路:包括染色體組裝,能量代謝,DNA損傷,氧化應(yīng)激和凋亡途徑。通過PKM-2驗證組學(xué)研究中的蛋白表達變化。此外,8a不僅抑制了HEX和PFK-1的蛋白表達,也顯著降低了乳酸的生成量。這些都證明糖酵解過程被8a顯著抑制。升高的XRCC6和降低的組蛋白也表明DNA損傷可能發(fā)生在8a作用的細胞中,而這些假設(shè)通過升高的γ-H2AX被證明。分子對接表明8a和DNA的相互作用方式包括3種氫鍵和4種π π相互作用,從而引起了DNA的損傷。第三,全細胞的糖組學(xué)分析一共鑒定出135種不同結(jié)構(gòu)的N糖和83種不同組成的N糖,其中有7種高甘露糖型的N糖在8a的刺激下呈現(xiàn)明顯降低的趨勢。細胞膜部分的糖組學(xué)分析一共鑒定出360種不同結(jié)構(gòu)的N糖和203種不同組成的N糖,其中一共有42種N糖在8a刺激前后呈現(xiàn)顯著性差異變化,這包括12種高甘露型,4種非修飾的復(fù)合/雜合型,9種巖藻糖型和9種唾液酸型N糖。相比起全細胞裂解液來說,細胞膜部分能更敏感的監(jiān)控吖啶酮藥物刺激下的糖基化變化。而這所有的糖基化變化被表明是和8a抑制了負責(zé)催化糖鏈轉(zhuǎn)移的酶DDOST有關(guān)。綜上所述,8a可能是通過下面多種機制來引發(fā)腫瘤細胞的死亡:氧化應(yīng)激調(diào)節(jié)的凋亡,能量途徑的紊亂,DNA的損傷等等。綜合以上多種組學(xué)手段的研究,不僅對8a的抗腫瘤作用機理提供了新穎的見解,也有助于加快8a結(jié)構(gòu)的進一步優(yōu)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 緯 -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.
【學(xué)位授予單位】：清華大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R96

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