NPM基因沉默提高淋巴细胞白血病细胞系CA46及Jurkat药物敏感性的初步研究

发布时间：2018-06-30 19:39

本文选题：白血病 + 多药耐药　；参考：《福建医科大学》2016年硕士论文

【摘要】：目的课题组前期通过蛋白质组学技术筛选出的核仁磷酸蛋白(Nucleophosmin,NPM,B23)在耐药的急性白血病患者中高表达,推测该蛋白的高表达可能与白血病的耐药机制相关。本课题旨在构建NPM基因稳定沉默的急性淋巴细胞白血病细胞系CA46及Jurkat细胞模型,研究沉默NPM后对CA46及Jurkat细胞株增殖、凋亡、药物敏感性等的影响,初步探讨NPM基因沉默对白血病细胞药物敏感性影响的可能机制。方法1.设计并构建针对NPM的小干扰RNA载体,包装慢病毒,分别转染CA46及Jurkat细胞,并检测其转染效率及干扰效率。2.应用MTT法和细胞克隆形成实验检测CA46及Jurkat细胞增殖变化。3.应用流式细胞术分析细胞凋亡,Western blot法研究凋亡相关蛋白的变化。4.应用MTT法检测NPM基因沉默后对白血病细胞阿霉素的敏感性(IC50),并用Western blot、实时荧光定量PCR技术法检测耐药相关蛋白及m RNA的表达水平。5.Western blot法研究若干信号通路的变化以探讨细胞凋亡及耐药的可能机制。6.本文中以CON、NC、KD分别表示CA46及Jurkat细胞的空白对照组、插入无关序列的阴性对照组和NPM基因敲减实验组。结果1.成功构建NPM基因沉默的慢病毒表达载体(NPM-RNAi-LV),并成功构建 NPM基因稳定沉默的CA46和Jurkat细胞株。2.NPM基因沉默能够导致CA46及Jurkat细胞增殖减慢,细胞克隆形成能力受抑制。3.NPM基因沉默能够诱导CA46及Jurkat细胞凋亡现象增加,其中caspase-3前体下调,剪切体上调;PARP剪切体上调;Bcl-2抗凋亡蛋白下调;CA46/KD组细胞中caspase-9剪切体上调;Jurkat/KD组细胞中caspase-9前体下调。4.NPM基因沉默提高CA46及Jurkat细胞对阿霉素的敏感性,抑制NPM表达后,CA46细胞对阿霉素的IC50值由转染前的0.526±0.138μg/ml下降至转染后的0.099±0.025μg/ml,敏感性提高5.313倍;Jurkat细胞对阿霉素的IC50值由转染前的0.528±0.098μg/ml下降至转染后的0.223±0.031μg/ml,敏感性提高2.368倍。5.NPM基因沉默后,BCRP、LRP表达下调,CA46和Jurkat细胞中AKT/m TOR的信号分子的活化受到抑制,c-Myc、GST-π基因的表达下调。Jurkat细胞中还出现p-ERK信号分子下调。结论RNA干扰成功下调CA46/Jurkat细胞株中NPM基因的表达,并证实NPM基因沉默可以导致CA46/Jurkat细胞增殖减慢,诱导细胞凋亡增加,caspase家族与Bcl-2家族参与细胞凋亡的调控。沉默NPM基因能够提高CA46/Jurkat细胞对阿霉素的敏感性,下调耐药相关蛋白BCRP、LRP的表达,可能与PI3K/AKT/m TOR信号通路的受抑制和c-Myc、GST-π基因表达下调有关。在Jurkat细胞中除上述作用机制外,还可能与p-ERK信号分子下调有关。
[Abstract]:Objective the high expression of Nucleophosmin (NPM, B23), which was screened by proteomics technology in the early stage, is highly expressed in the drug resistant acute leukemia patients. It is presumed that the high expression of the protein may be related to the mechanism of drug resistance in leukemia. The aim of this study is to construct a stable and silent NPM gene of acute lymphoblastic leukemia cell line CA46. And Jurkat cell model, the effects of silent NPM on the proliferation, apoptosis and drug sensitivity of CA46 and Jurkat cell lines were investigated. The possible mechanism of NPM gene silencing on the drug sensitivity of leukemia cells was preliminarily discussed. Method 1. the small interference RNA vector for NPM was designed and constructed, the lentivirus was packaged, CA46 and Jurkat cells were transfected respectively, and the detection of CA46 and Jurkat cells respectively. Test the transfection efficiency and interference efficiency.2. MTT method and cell clone formation test to detect CA46 and Jurkat cell proliferation changes.3. application flow cytometry analysis of apoptosis, Western blot method to study the changes of apoptosis related proteins.4. application MTT method to detect the sensitivity of adriamycin in leukemic cells after NPM gene silencing (IC50), and Weste RN blot, real-time fluorescence quantitative PCR technique was used to detect the expression level of drug-resistant related proteins and m RNA by.5.Western blot method to study the changes of several signaling pathways to explore the possible mechanism of cell apoptosis and drug resistance.6. in this paper, CON, NC, KD, respectively, expressed in the group of CA46 and blank cells, the negative control group inserted into the independent sequence and the negative control group, respectively. The result of gene knockout experimental group. Results 1. the NPM gene silenced Lentivirus Expression Vector (NPM-RNAi-LV) was successfully constructed, and the successful construction of the silent CA46 and.2.NPM gene silencing of the NPM gene could lead to the proliferation and decrease of CA46 and Jurkat cells, and the cell clone formation ability was inhibited by the suppression of.3.NPM gene silencing, which could induce CA46 and Jurkat fine. The apoptosis was increased, in which the caspase-3 precursor was down, the shear body was up regulation, the PARP shear body was up regulated, the Bcl-2 anti apoptotic protein was down regulated, the caspase-9 shear body was up regulated in the CA46/KD group, and the caspase-9 precursor in the Jurkat/KD group reduced the.4.NPM gene silencing to increase the CA46 and Jurkat cell sensitivity to doxorubicin, and the CA46 cells were inhibited after the NPM expression. The IC50 value of adriamycin decreased from 0.526 + 0.138 g/ml before transfection to 0.099 + 0.025 mu g/ml after transfection, and the sensitivity was increased by 5.313 times. The IC50 value of adriamycin in Jurkat cells decreased from 0.528 + 0.098 Mu before transfection to 0.223 + 0.031 g/ml after transfection, and the sensitivity was increased by 2.368 times.5.NPM gene, BCRP, LRP expression was down, CA46 and Jur The activation of signal molecules of AKT/m TOR in Kat cells was inhibited, and the expression of c-Myc, GST- PI gene was downregulated in.Jurkat cells. Conclusion RNA interference successfully downregulated the expression of NPM gene in CA46/Jurkat cell lines, and confirmed that NPM gene silencing could lead to the proliferation and decrease of CA46 cells and induce cell apoptosis. The caspase family and the Bcl-2 family are involved in the regulation of apoptosis. The silence of NPM gene can improve the sensitivity of CA46/Jurkat cells to adriamycin, and the expression of BCRP, LRP, may be related to the inhibition of PI3K/AKT/m TOR signaling pathway and the regulation of c-Myc, GST- PI gene expression. It may also be related to the downregulation of p-ERK signal molecules.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.71

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