锑剂治疗急性早幼粒细胞白血病的优势作用研究

发布时间：2018-02-28 07:49

本文关键词： 三氧化二砷三氧化二锑锑化合物分化砷剂锑剂　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的：1)研究锑剂在治疗急性早幼粒细胞白血病(APL)中的作用,探究锑剂对急性早幼粒细胞白血病相关蛋白的影响,进而证明锑剂具有治疗APL作用。2)通过探讨锑剂对PML突变体(例如A216V和L218P；砷剂耐药性突变体)的降解作用,发现具有更强降解PML突变体的锑化合物。方法：常规培养急性早幼粒细胞白血病NB4细胞后,使用三氧化二锑(简称锑剂)或砷剂(阳性对照)暴露NB4细胞,通过MTT法,瑞氏-吉姆萨染料染色,流式细胞术,Western Blot,免疫荧光等方法检测NB4细胞的增值抑制,分化、凋亡等指标变化。使用免疫荧光法检测锑剂对核小体形成的影响。利用HEK293T或HeLa细胞,转染Flag-PML或Flag-PML-RARa基因,过表达PML或PML-RARa融合蛋白,研究锑剂(或砷剂)对PML蛋白的溶性改变和降解作用。主要以RIPA buffer和LDS buffer分别提取可溶上清蛋白(RIPA可溶部分)和不溶沉淀蛋白(RIPA不可溶部分)。使用Western Blot法分别检测PML和PML-RARa融合蛋白在RIPA可溶和不溶部分中的变化和后修饰变化。结果：1)锑剂对NB4细胞的作用结果；MTT和流式细胞术结果显示,锑剂的细胞毒性明显弱于砷剂,而且锑剂有较强的诱导NB4细胞CD11b的表达(早有粒细胞分化标志蛋白)。同时,还发现锑剂可使NB细胞核形态变化,证明锑剂具有较强的诱导NB4细胞分化作用。随着锑剂暴露浓度的增加,胞形态出现了凋亡的特征,证明锑剂具有一定的诱导细胞凋亡的作用。Western Blot和免疫荧光共聚焦结果显示,锑剂同样可导致PML-RARa融合蛋白的溶性发生改变,最终降解其蛋白融合蛋白；而且出现大颗粒状的PML核小体,并且与SUMO-1共定位于细胞核,提示锑剂可促使PML-RARα融合蛋白进入PML核小体,进而促进融合蛋白的降解,其作用类似于砷剂。2)锑剂对PML或PML-RARa过表达293T细胞体系实验结果；分别用不同浓度的锑剂或不同时间处理PML和PML-RARa过表达293T细胞,锑剂同样能够改变PML或PML-RARa融合蛋白的溶性改变,与NB4细胞实验结果相吻合。3)锑剂和PSO(锑化合物)对PML突变体的作用结果；锑剂和砷剂都无法诱导PML突变体(A216V或L218P)的溶性改变或降解,而新型锑化合物PSO具有较强的诱导PML突变体的溶性发生改变。结论：锑剂具有类似于砷剂诱导急性早幼粒细胞白血病NB4细胞的分化的作用,并且锑剂的细胞毒性远低于比砷剂。进一步证明锑剂同样能够诱导PML和PML-RARα融合蛋白的溶性改变和降解其融合蛋白。锑剂和砷剂对PML突变体无作用,而新型锑化合物PSO具有较强的诱导砷剂耐药型PML突变蛋白降解,从而证明锑剂作用的广泛性。
[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.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.71

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