【摘要】：急性髓系白血病(Acute myeloid leukemia,AML)是造血干/祖細(xì)胞起源的一組異質(zhì)性克隆性腫瘤,目前臨床上對AML病人的標(biāo)準(zhǔn)聯(lián)合化療方案存在諸多問題。熱休克蛋白(Hsp)90在AML中高表達(dá),在細(xì)胞內(nèi)起到維護(hù)癌蛋白穩(wěn)定的作用,使細(xì)胞最終惡性轉(zhuǎn)化為白血病細(xì)胞。研究表明Hsp90可作為靶向治療AML的良好靶點。本項目研究新型Hsp90抑制劑SNX-2112抗人急性骨髓系白血病(AML)細(xì)胞的活性。首先,采用CCK8檢測SNX-2112對兩株AML細(xì)胞株增殖的影響;然后通過流式細(xì)胞術(shù)和western blot檢測SNX-2112對AML細(xì)胞的細(xì)胞周期和凋亡影響;并且通過流式細(xì)胞術(shù)檢測分化相關(guān)表面標(biāo)記物CD11b的表達(dá),western blot和qRT-PCR,軟瓊脂克隆形成以及瑞氏-吉姆薩染色等方法用于探究SNX-2112對兩株細(xì)胞分化的影響;最后采用western blot和激光共聚焦來初步的分析和觀察SNX-2112作用于AML細(xì)胞的作用機(jī)制。結(jié)果表明,SNX-2112能夠顯著的抑制AML細(xì)胞的增殖,且效果要較經(jīng)典的Hsp90抑制劑-17-AAG顯著,對人正常細(xì)胞的細(xì)胞毒性較小;SNX-2112能誘導(dǎo)AML細(xì)胞的G2/M期阻滯,且下調(diào)周期蛋白A的表達(dá),同時SNX-2112能誘導(dǎo)AML細(xì)胞發(fā)生凋亡,引起凋亡相關(guān)蛋白caspase3等的激活;此外SNX-2112可誘導(dǎo)AML細(xì)胞的分化,抑制原癌基因C-Myc的表達(dá),并上調(diào)分化相關(guān)轉(zhuǎn)錄因子PU.1、C/EBPα的表達(dá);機(jī)制研究表明,SNX-2112抑制了Hsp90的客戶蛋白IKK、Akt的表達(dá),并抑制相關(guān)下游信號通路的激活。結(jié)論:本研究表明了SNX-2112體外誘導(dǎo)AML周期阻滯、凋亡以及分化,具有抗急性髓性白血病的潛在效果,其作用機(jī)制可能是通過抑制PI3K/NF-κB信號通路的激活,下調(diào)c-Myc的表達(dá),激活分化相關(guān)轉(zhuǎn)錄因子的表達(dá)。本研究為SNX-2112治療急性髓性白血病奠定了研究基礎(chǔ),為SNX-2112的臨床前應(yīng)用提供理論研究。
[Abstract]:Acute myeloid leukemia (Acute myeloid leukemia,AML) is a heterogeneous clonal tumor of hematopoietic stem / progenitor cell origin. At present, there are many problems in the standard combination chemotherapy regimen for AML patients. Heat shock protein (Hsp) 90 (HSP90) is highly expressed in AML and plays a role in maintaining the stability of oncoprotein in the cells, which leads to the malignant transformation of the cells into leukemia cells. Studies have shown that Hsp90 can be used as a good target for targeted treatment of AML. The aim of this study was to investigate the activity of SNX-2112, a novel Hsp90 inhibitor, against human acute myeloid leukemia (AML) cells. Firstly, the effects of SNX-2112 on the proliferation of two AML cell lines were detected by CCK8, then the effects of SNX-2112 on the cell cycle and apoptosis of AML cells were detected by flow cytometry and western blot. The expression of CD11b, western blot and qRT-PCR, soft Agar clone formation were detected by flow cytometry, and the effects of SNX-2112 on the differentiation of two cell lines were investigated by Ricker-Gimsa staining. Finally, western blot and laser confocal focus were used to analyze and observe the mechanism of SNX-2112 acting on AML cells. The results showed that SNX-2112 could significantly inhibit the proliferation of AML cells, and the effect was more significant than that of classical Hsp90 inhibitor-17-AAG, and the cytotoxicity to human normal cells was less. SNX-2112 could induce G _ 2 / M arrest and down-regulate the expression of cyclin A in AML cells, and SNX-2112 could induce apoptosis of AML cells and induce the activation of apoptosis-related protein caspase3. In addition, SNX-2112 could induce the differentiation of AML cells, inhibit the expression of proto-oncogene C-Myc and up-regulate the expression of differentiation related transcription factor PU.1,C/EBP 偽. SNX-2112 inhibits the expression of Hsp90 client protein IKK,Akt and inhibits the activation of related downstream signaling pathways. Conclusion: this study suggests that SNX-2112 induces AML cycle arrest, apoptosis and differentiation in vitro, which has the potential effect of anti-acute myeloid leukemia, and its mechanism may be by inhibiting the activation of PI3K/NF- 魏 B signaling pathway. The expression of c-Myc was down-regulated and the expression of differentiation related transcription factors was activated. This study lays a foundation for the treatment of acute myeloid leukemia with SNX-2112 and provides a theoretical study for the preclinical application of SNX-2112.
【學(xué)位授予單位】：暨南大學(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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