【摘要】：混合譜系白血病(Mixed Lineage Leukemia,MLL),因11q23處MLL基因易位重排形成融合蛋白而得名,是一類致死率極高、預(yù)后差的高危惡性急性白血病。DOT1L蛋白是目前發(fā)現(xiàn)的唯一H3K79甲基化轉(zhuǎn)移酶,也是MLL白血病的重要靶點(diǎn),開發(fā)DOT1L小分子抑制劑成為治療MLL白血病的一條新思路。多胺類化合物也因其多靶點(diǎn)性成為抗腫瘤藥物的研究熱點(diǎn),目前對(duì)于DOT1L抑制劑的設(shè)計(jì)也主要為多胺類衍生物。本課題首先建立組蛋白甲基轉(zhuǎn)移酶DOT1L抑制劑的虛擬篩選方法,篩選出與DOT1L受體對(duì)接評(píng)分較高的化合物;設(shè)計(jì)合成路線合成、分離純化目標(biāo)化合物,并進(jìn)行結(jié)構(gòu)確認(rèn);最后考察目標(biāo)化合物對(duì)混合譜系白血病細(xì)胞的體外活性研究。研究結(jié)果如下:(1)根據(jù)實(shí)驗(yàn)室前期研究基礎(chǔ)結(jié)合相關(guān)文獻(xiàn)資料,設(shè)計(jì)一系列多胺類衍生物,與正處于一期臨床實(shí)驗(yàn)的陽性化合物EPZ-5676共同構(gòu)成配體化合物庫,從蛋白質(zhì)晶體數(shù)據(jù)庫PDB中選取受體模型4HRA。用Sybyl-X2.0軟件將受體與配體進(jìn)行分子對(duì)接試驗(yàn),篩選出了分?jǐn)?shù)最高的化合物DUOA003作為目標(biāo)化合物;(2)設(shè)計(jì)合成路線并完成目標(biāo)化合物DUOA003的合成工作,通過LC-MS、1HNMR,13CNMR確定目標(biāo)化合物的結(jié)構(gòu)為:N,N'-(propane-1,3-diyl)bis(2,5-dihydroxybenzamide);(3)以混合譜系白血病細(xì)胞MV4-11和巨噬細(xì)胞作為研究對(duì)象,采用CCK-8法測(cè)定兩株細(xì)胞在目標(biāo)化合物和陽性藥物阿糖胞苷不同濃度(120、100、50、25、10、1、0.1μM)作用24h后的增殖抑制率。結(jié)果發(fā)現(xiàn)DUOA003和陽性藥物阿糖胞苷對(duì)MV4-11細(xì)胞均有顯著的抑制作用,IC50分別為25和28μM,對(duì)巨噬細(xì)胞的IC50分別為71μM和11μM;選擇對(duì)正常細(xì)胞安全濃度范圍考察不同作用時(shí)間24h、48h、72h對(duì)細(xì)胞抑制作用的影響,結(jié)果反映出DUOA003對(duì)MV4-11細(xì)胞的抑制率展現(xiàn)出時(shí)間和劑量的依賴性;(4)采用Annexin V-FITC/PI法對(duì)經(jīng)高、中、低濃度的藥物誘導(dǎo)24h和48h的MV4-11細(xì)胞進(jìn)行染色,在熒光顯微鏡下觀察誘導(dǎo)48h后細(xì)胞狀態(tài),并用流式細(xì)胞儀檢測(cè)目標(biāo)化合物誘導(dǎo)MV4-11細(xì)胞的凋亡率。結(jié)果發(fā)現(xiàn)DUOA003能夠顯著地誘導(dǎo)MV4-11細(xì)胞產(chǎn)生凋亡。誘導(dǎo)48h后,與陰性對(duì)照組(17.3±5%)相比,DUOA003的低濃度組(53.9±8%)、中濃度組(80.9±9%)、高濃度組(88.5±6%)、以及阿糖胞苷高濃度組(82.6±9%)均呈現(xiàn)顯著性差異(P0.001),DUOA003誘導(dǎo)的細(xì)胞48h后凋亡多處于晚期,阿糖胞苷誘導(dǎo)的細(xì)胞凋亡則主要出現(xiàn)在早期;(5)用Caspase-3活性測(cè)定試劑盒,檢測(cè)經(jīng)高、中、低濃度的目標(biāo)化合物誘導(dǎo)細(xì)胞凋亡24h后Caspase-3的活性變化。Caspase-3活性檢測(cè)顯示化合物DUOA003在誘導(dǎo)細(xì)胞凋亡24后,與陰性對(duì)照組Caspase-3活性測(cè)定OD值(0.146±0.002)比較,DUOA003的低濃度組(0.227±0.003)、中濃度組(0.367±0.004)、高濃度組(0.554±0.005)以及阿糖胞苷高濃度組(0.477±0.006)均呈現(xiàn)顯著性差異(P0.001)。DUOA003誘導(dǎo)混合譜系白血病細(xì)胞MV4-11凋亡過程中存在Caspase-3介導(dǎo)的細(xì)胞凋亡。
[Abstract]:Mixed lineage leukemia (Mixed Lineage Leukemia,MLL), named after MLL gene translocation and fusion protein in 11q23, is the only H3K79 methyltransferase found in high risk acute leukemia patients with high mortality and poor prognosis. It is also an important target of MLL leukemia. The development of small molecular inhibitors of DOT1L has become a new idea in the treatment of MLL leukemia. Polyamines have become the research focus of antitumor drugs because of their multi-target, and the current design of DOT1L inhibitors is mainly polyamine derivatives. Firstly, a virtual screening method of histone methyltransferase DOT1L inhibitor was established to screen the compounds with high docking score with DOT1L receptor, then the synthesis route was designed, the target compounds were isolated and purified, and the structure of the target compounds was confirmed. Finally, the in vitro activity of the target compounds to mixed lineage leukemia cells was investigated. The results are as follows: (1) A series of polyamines derivatives were designed according to the basis of pre-laboratory research and related literature, and the ligands library was constructed together with the positive compound EPZ-5676, which is in the primary clinical trial. The receptor model 4HRAwas selected from protein crystal database PDB. Molecular docking test between receptor and ligand was carried out by Sybyl-X2.0 software and the highest fraction compound DUOA003 was selected as the target compound. (2) the synthetic route was designed and the synthesis of target compound DUOA003 was completed. The structure of the target compound was determined by LC-MS,1HNMR,13CNMR to be propane-1,3-diyl) bis (2-dihydroxybenzamide); (3. The mixed lineage leukemic cells MV4-11 and macrophages were used as the research objects. CCK-8 assay was used to determine the proliferation inhibition rate of the two cell lines at different concentrations of target compound and cytarabine at different concentrations (120 ~ 100g / 100) for 24 hours after exposure to 0.1 渭 M of cytosine arabinoside, the target compound and the positive drug cytarabine. The results showed that DUOA003 and cytarabine had significant inhibitory effects on MV4-11 cells, IC50 were 25 渭 M and 28 渭 M, and IC50 on macrophages were 71 渭 M and 11 渭 M, respectively. The effect of cell inhibition, The results showed that the inhibition rate of DUOA003 on MV4-11 cells was time-and dose-dependent. (4) Annexin V-FITC/PI method was used to stain the MV4-11 cells induced by high, medium and low concentrations of drugs for 24 and 48 hours, and the cells were observed under fluorescence microscope after 48 hours of induction. The apoptosis rate of MV4-11 cells induced by target compounds was detected by flow cytometry. The results showed that DUOA003 could induce apoptosis of MV4-11 cells significantly. 48 h after induction, there were significant differences between the low concentration group (53.9 鹵8%), the middle concentration group (80.9 鹵9%), the high concentration group (88.5 鹵6%) and the high concentration group of cytarabine (82.6 鹵9%) compared with the negative control group (17.3 鹵5%). The apoptosis induced by cytarabine mainly occurred in the early stage. (5) the Caspase-3 activity assay kit was used to detect the cell apoptosis. The changes of Caspase-3 activity after 24 hours of apoptosis induced by low concentration of target compound. The activity of Caspase-3 showed that the compound DUOA003 induced apoptosis 24 hours later. Compared with the negative control group (0.146 鹵0.002), the low concentration group (0.227 鹵0.003), the middle concentration group (0.367 鹵0.004), the high concentration group (0.554 鹵0.005) and the high concentration group (0.477 鹵0.006) showed significant difference in the process of MV4-11 apoptosis induced by DUOA003. There is Caspase-3 mediated apoptosis.
【學(xué)位授予單位】：重慶理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R733.7

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