本文關(guān)鍵詞： 三氧化二砷 三氧化二銻 銻化合物 分化 砷劑 銻劑　出處：《浙江大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：1)研究銻劑在治療急性早幼粒細(xì)胞白血病(APL)中的作用,探究銻劑對急性早幼粒細(xì)胞白血病相關(guān)蛋白的影響,進(jìn)而證明銻劑具有治療APL作用。2)通過探討銻劑對PML突變體(例如A216V和L218P；砷劑耐藥性突變體)的降解作用,發(fā)現(xiàn)具有更強降解PML突變體的銻化合物。方法：常規(guī)培養(yǎng)急性早幼粒細(xì)胞白血病NB4細(xì)胞后,使用三氧化二銻(簡稱銻劑)或砷劑(陽性對照)暴露NB4細(xì)胞,通過MTT法,瑞氏-吉姆薩染料染色,流式細(xì)胞術(shù),Western Blot,免疫熒光等方法檢測NB4細(xì)胞的增值抑制,分化、凋亡等指標(biāo)變化。使用免疫熒光法檢測銻劑對核小體形成的影響。利用HEK293T或HeLa細(xì)胞,轉(zhuǎn)染Flag-PML或Flag-PML-RARa基因,過表達(dá)PML或PML-RARa融合蛋白,研究銻劑(或砷劑)對PML蛋白的溶性改變和降解作用。主要以RIPA buffer和LDS buffer分別提取可溶上清蛋白(RIPA可溶部分)和不溶沉淀蛋白(RIPA不可溶部分)。使用Western Blot法分別檢測PML和PML-RARa融合蛋白在RIPA可溶和不溶部分中的變化和后修飾變化。結(jié)果：1)銻劑對NB4細(xì)胞的作用結(jié)果；MTT和流式細(xì)胞術(shù)結(jié)果顯示,銻劑的細(xì)胞毒性明顯弱于砷劑,而且銻劑有較強的誘導(dǎo)NB4細(xì)胞CD11b的表達(dá)(早有粒細(xì)胞分化標(biāo)志蛋白)。同時,還發(fā)現(xiàn)銻劑可使NB細(xì)胞核形態(tài)變化,證明銻劑具有較強的誘導(dǎo)NB4細(xì)胞分化作用。隨著銻劑暴露濃度的增加,胞形態(tài)出現(xiàn)了凋亡的特征,證明銻劑具有一定的誘導(dǎo)細(xì)胞凋亡的作用。Western Blot和免疫熒光共聚焦結(jié)果顯示,銻劑同樣可導(dǎo)致PML-RARa融合蛋白的溶性發(fā)生改變,最終降解其蛋白融合蛋白；而且出現(xiàn)大顆粒狀的PML核小體,并且與SUMO-1共定位于細(xì)胞核,提示銻劑可促使PML-RARα融合蛋白進(jìn)入PML核小體,進(jìn)而促進(jìn)融合蛋白的降解,其作用類似于砷劑。2)銻劑對PML或PML-RARa過表達(dá)293T細(xì)胞體系實驗結(jié)果；分別用不同濃度的銻劑或不同時間處理PML和PML-RARa過表達(dá)293T細(xì)胞,銻劑同樣能夠改變PML或PML-RARa融合蛋白的溶性改變,與NB4細(xì)胞實驗結(jié)果相吻合。3)銻劑和PSO(銻化合物)對PML突變體的作用結(jié)果；銻劑和砷劑都無法誘導(dǎo)PML突變體(A216V或L218P)的溶性改變或降解,而新型銻化合物PSO具有較強的誘導(dǎo)PML突變體的溶性發(fā)生改變。結(jié)論：銻劑具有類似于砷劑誘導(dǎo)急性早幼粒細(xì)胞白血病NB4細(xì)胞的分化的作用,并且銻劑的細(xì)胞毒性遠(yuǎn)低于比砷劑。進(jìn)一步證明銻劑同樣能夠誘導(dǎo)PML和PML-RARα融合蛋白的溶性改變和降解其融合蛋白。銻劑和砷劑對PML突變體無作用,而新型銻化合物PSO具有較強的誘導(dǎo)砷劑耐藥型PML突變蛋白降解,從而證明銻劑作用的廣泛性。
[Abstract]:Objective: 1) of antimony agent in the treatment of acute promyelocytic leukemia (APL) in the role, to explore the impact of antimonials on acute promyelocytic leukemia related protein, and prove that APL.2 treatment agent with antimony antimony) through the study of mutant agent on PML (such as A216V and L218P; arsenic resistance mutant) the degradation effect of antimony compounds found has stronger degradation of PML mutants. Methods: acute promyelocytic leukemia NB4 cells, using three oxidation two antimony (the antimony agent) or arsenic trioxide (positive control) exposure of NB4 cells by MTT method, Wright Giemsa dye staining and flow cytometry. Western Blot, NB4 cell proliferation inhibition test, immunofluorescence methods such as differentiation, apoptosis index. Effects of antimony detection agent on nucleosome formation using immunofluorescence method. By using HEK293T or HeLa cells transfected with Flag-PML or Flag-PML-RARa Because, over expression of PML or PML-RARa fusion protein of antimonials (or arsenic) on PML protein solubility change and degradation. The main RIPA buffer and LDS buffer respectively to extract soluble supernatant protein (RIPA soluble fraction) and insoluble protein precipitation (RIPA insoluble part). Using the Western Blot method was used to detect PML PML-RARa and RIPA fusion protein in soluble and insoluble part of the change and modification changes. Results: 1) the effect of antimony agent on NB4 cells; MTT and flow cytometry showed that the cytotoxicity of antimonials was weaker than that of arsenic, antimony and agents have strong NB4 cells induced the expression of CD11b (early myeloid differentiation marker). At the same time, also found that antimonials can make NB morphological changes, that antimonials have strong NB4 cell differentiation effect. With the increase of antimony exposure concentration, cell morphology appeared apoptosis characteristics of proof agent with sb There are some apoptosis inducing effects of.Western Blot and confocal immunofluorescence results showed that antimonials can result in PML-RARa fusion protein solubility change, the final degradation of fusion proteins; and large granular PML nucleosomes, and SUMO-1 colocalized in the nucleus, suggesting that antimonials can promote PML-RAR alpha fusion protein PML into the nucleosome, and then promote the degradation of the fusion protein, its role is similar to arsenic antimony.2) agent on PML or PML-RARa cells overexpressing 293T system experimental results; respectively with different concentrations of antimony agents or different processing time of PML and PML-RARa cells overexpressing 293T, antimonials can also change the PML or PML-RARa fusion protein solubility change of NB4 cell and the experimental results were in good agreement with.3) and PSO (antimony antimony compounds) effect on results of PML mutant; antimony and arsenic trioxide can induce agent PML mutants (A216V or L2 18P) the solubility change or degradation, while the new antimony compound PSO has strong induction of PML mutant soluble change. Conclusion: Antimony is similar to arsenic induced acute promyelocytic leukemia NB4 cell differentiation, and cell toxicity of antimonials is far lower than the ratio of arsenic. Further proof of the same antimonials could induce PML and PML-RAR alpha fusion protein solubility change and degradation of its fusion protein. Antimony and arsenic trioxide had no effect on PML mutant, and new antimony compound PSO is induced by arsenic trioxide resistant PML strong mutant protein degradation, thus proving extensive antimonials.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R733.71

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