DBO诱导K562细胞凋亡与自噬的作用研究

发布时间：2018-06-30 20:11

本文选题：DBO + K562细胞　；参考：《济南大学》2017年硕士论文

【摘要】：背景慢性髓系细胞白血病(CML)是一种起源于多能干细胞的髓系恶性增殖性肿瘤,t(9;22)(q34;q11)是CML特征性染色体改变并在分子水平上导致BCR-ABL融合基因形成的。这种BCR-ABL融合基因形成的融合蛋白是一种能导致细胞转化的多种信号蛋白相关的酪氨酸激酶。目前酪氨酸激酶抑制剂(TKI)(包括伊马替尼,格列卫等)已广泛用于CML治疗,使大部分CML患者生存期延长,达到细胞遗传学或分子生物学上的缓解。随着TKI临床应用时间延长,TKI耐药的问题也越来越明显,积极探索新的药物是一个亟待解决的问题。DBO(6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine)是一种苯并恶嗪衍生物,有研究证实其可作为哺乳动物雷帕霉素靶蛋白(m TOR)抑制剂诱导人脐静脉内皮细胞产生细胞活性氧物质(ROS),从而诱导人脐静脉内皮细胞发生自噬,促进其凋亡。然而,目前其抗肿瘤作用尚未广泛研究,抗肿瘤作用的机制目前尚无定论。关于DBO是否引起白血病细胞发生自噬、凋亡的研究尚未报道。本研究以人K562细胞为研究对象,分析DBO对K562细胞的增殖及其在诱导细胞凋亡和自噬方面的影响。目的本研究旨在探讨DBO对人慢性髓系白血病K562细胞的增殖、自噬与凋亡的影响。方法常规方法复苏、传代培养K562细胞,设置对照组(DMSO)、DBO处理组。处理24、48和72h后收集细胞,采用蛋白质印迹法检测LC3蛋白的表达。细胞免疫荧光实验检测LC3斑点的表达;透射电镜观察细胞自噬现象。CCK-8比色法检测K562细胞的活性和增殖能力。细胞周期实验检测K562细胞分裂能力。Annexin V-FITC/PI双染流式细胞术检测细胞凋亡。结果蛋白质印迹结果显示,K562细胞经雷帕霉素处理后LC3-Ⅱ蛋白表达水平升高,p62蛋白表达水平降低;经50μmol/L DBO处理24、48和72 h后,LC3-Ⅱ和p62蛋白表达水平与雷帕霉素处理后结果相似,并呈时间依赖性。K562细胞经10、25和50μmol/L DBO处理后,LC3-Ⅱ蛋白表达水平升高,p62蛋白表达水平降低,呈剂量依赖性,而使用自噬抑制剂3-MA后LC3-Ⅱ蛋白表达降低,P62蛋白表达升高。细胞免疫荧光实验显示,DBO作用72 h后,与对照组相比,DBO处理组LC3斑点的表达量增加。透射电镜观察结果显示,与对照组相比,DBO处理组细胞胞质内可见较多自噬小体和自噬溶酶体,细胞核不规则,染色质边缘化。CCK8检测结果显示,DBO对K562细胞的增殖具有明显的抑制作用,并呈现时间-剂量依赖性。DBO经不同浓度(10μmol/L、25μmol/L、50μmol/L、100μmol/L)处理24 h的细胞增殖抑制率分别为(0.68±0.05)%、(2.76±0.35)%、(12.64±3.90)%、(22.58±2.41)%,F=67.389,P0.001,处理组与对照组之间差异有统计学意义;48 h的增殖抑制率分别为(3.83±1.06)%、(6.23±1.27)%、(15.90±1.10)%、(24.50±2.51)%,F=145.738,P0.001,处理组与对照组之间差异有统计学意义;72h的增殖抑制率分别为(8.78±1.28)%、(21.38±1.47)%、(32.11±2.01)%、(34.27±2.59)%,F=225.820,P0.001,处理组与对照组之间差异有统计学意义。流式细胞术分析显示K562细胞经DBO处理72 h后,与对照组相比,G2/M期细胞比例增加,χ2=276.706,P0.001。流式细胞术分析显示,与对照组相比,K562细胞经DBO处理72 h后的凋亡率增加,χ2=227.384,P0.001。结论DBO可明显抑制K562细胞生长,并与自噬和凋亡的发生有关。
[Abstract]:Background chronic myelocytic leukemia (CML) is a malignant proliferative tumor of myeloid origin derived from pluripotent stem cells. T (9; 22) (q34; Q11) is a characteristic chromosome change of CML and causes the formation of BCR-ABL fusion genes at the molecular level. The fusion protein formed by the BCR-ABL fusion gene is a variety of signal eggs that can lead to cell transformation. White related tyrosine kinase. Currently, tyrosine kinase inhibitors (TKI) (including imatinib, gleevet, etc.) have been widely used in CML treatment to prolong the survival of most CML patients and achieve cytogenetic or molecular biological remission. With the prolongation of the time of TKI clinical application, the problem of TKI resistance is becoming more and more obvious, actively exploring new Drug is an urgent problem.DBO (6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine) is a benzoxazine derivative. It has been proved that it can induce human umbilical vein endothelial cells to produce fine cell active oxygen substance (ROS) as a target protein of mammalian rapamycin target protein (m TOR) and induce the human umbilical vein endothelium. However, the antitumor effect of DBO has not been widely studied, and the mechanism of anti-tumor effect is not yet conclusive. The study of whether it causes autophagy and apoptosis in leukemia cells has not been reported. This study is based on the study of human K562 cells and the analysis of the proliferation of K562 cells by DBO and its induced cells. The effect of apoptosis and autophagy. The purpose of this study was to explore the effect of DBO on the proliferation, autophagy and apoptosis of human chronic myeloid leukemia K562 cells. Methods routine methods of resuscitation, generation of K562 cells, control group (DMSO), DBO treatment group, 24,48 and 72h were used to collect cells, and the expression of LC3 protein was detected by Western blot. Cell immunofluorescence test was used to detect the expression of LC3 spots; transmission electron microscopy was used to observe the cell autophagy by.CCK-8 colorimetric assay to detect the activity and proliferation of K562 cells. Cell cycle test was used to detect K562 cell mitosis by.Annexin V-FITC/PI double dye flow cytometry to detect cell apoptosis. Results of Western blot showed that K562 cells were Rima. The expression level of LC3- II protein increased and the expression level of p62 protein decreased. After treatment of 24,48 and 72 h by 50 mol/L DBO, the expression level of LC3- II and p62 protein was similar to that after the treatment of rapamycin, and the time dependent.K562 cells were treated with 10,25 and 50 mu mol/L DBO, and the protein expression level was increased. After the use of autophagy inhibitor 3-MA, the expression of LC3- II protein decreased and the expression of P62 protein increased. The cell immunofluorescence test showed that the expression of LC3 spots in the DBO treatment group increased after 72 h, compared with the control group. The results of transmission electron microscopy showed that in the cytoplasm of the DBO treatment group compared with the control group, the cytoplasm of the DBO treatment group was visible. More autophagosomes and autophagosomes, nuclei are irregular, and chromatin marginalization.CCK8 detection results show that DBO has a significant inhibitory effect on the proliferation of K562 cells, and the proliferation inhibition rate of time dose dependent.DBO by different concentrations (10 mu mol/L, 25 mu mol/L, 50 mu mol/L, 100 mu mol/L) is (0.68 + 0.05)% respectively (0.68 + 0.05)%. (2.76 + 0.35)%, (12.64 + 3.90)%, (22.58 + 2.41)%, F=67.389, P0.001, the difference between the treatment group and the control group was statistically significant, the proliferation inhibition rate of 48 h was (3.83 + 1.06)%, (6.23 + 1.27)%, (15.90 + 1.10)%, (15.90 + 1.10)%, F=, P0.001, and the difference between the treatment group and the control group was statistically significant; the proliferation inhibition rate of 72h was respectively, respectively. (8.78 + 1.28)%, (21.38 + 1.47)%, (32.11 + 2.01)%, (34.27 + 2.59)%, F=225.820, P0.001, the difference between the treatment group and the control group was statistically significant. The flow cytometry analysis showed that after DBO treatment 72 h, compared with the control group, the G2/M phase cells were increased compared with the control group, and the Chi 2=276.706, P0.001. flow cytometry analysis showed that compared with the control group, the analysis showed that the cells were compared with the control group. The apoptotic rate of K562 cells treated with DBO after 72 h treatment increased, 2=227.384 P0.001.. Conclusion DBO can significantly inhibit the growth of K562 cells, and is related to the occurrence of autophagy and apoptosis.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.72

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