新甾醇类药物NSC67657诱导白血病细胞HL-60单核系分化及其机制研究

发布时间：2018-02-02 16:30

本文关键词： NSC67657 白血病细胞单核系分化 ICAT Wnt/β-catenin信号通路　出处：《重庆医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的构建新甾醇类药物NSC6767诱导白血病细胞HL-60向单核系分化的细胞模型,检测药物对细胞的生物学行为的影响、探讨其可能的分子机制。方法以10μmol/L NSC67657药物处理HL-60细胞5 d构建细胞分化模型,以DMSO溶剂处理HL-60细胞5 d为对照组;采用瑞氏染色、碱性α-丁酸萘酚酯酶(α-NBE)染色分析细胞的分化方向;采用流式细胞术分析细胞的分化程度、细胞周期分布、细胞凋亡的情况;采用乳酸脱氢酶(LD)检测试剂盒检测药物对细胞的毒性作用;采用RT-PCR和Western blot分析ICAT、β-catenin、T细胞因子4(TCF-4)的m RNA和蛋白在细胞分化前后的表达情况;采用Western blot和免疫荧光染色方法检测细胞分化前后β-catenin,TCF-4在细胞内定位情况。采用RT-PCR和Western blot分析Wnt信号通路下游靶基因c-myc、细胞周期蛋白D1(cyclin D1)、TCF-1 m RNA和蛋白在细胞分化前后的表达情况;结果瑞氏染色后观察发现:细胞在10μmol/L NSC67657作用5 d后细胞质明显增多,核仁消失,细胞核呈圆形或不规则形;α-NBE染色结果显示:对照组胞质无色,为阴性,实验组胞质中出现鲜红色的颗粒沉淀,为阳性,并能被氟化钠(Na F)抑制,抑制率50%。流式细胞术(FCM)结果显示:10μmol/L NSC67657作用5 d后的实验组CD14+细胞数为(94.37±2.84)%,高于对照组[(1.31±0.09)%],差异有统计学意义(P0.01);实验组G1/G0期细胞所占比例为(76.46±2.83)%,高于对照组[(59.4±5.42)%],差异有统计学意义(P0.05);实验组S期细胞所占比例为(18.76±0.98)%,低于对照组[(34.38±2.61)%],差异有统计学意义(P0.05);药物处理前后的HL-60细胞早期和晚期凋亡均无明显改变(均P0.05);LD检测结果显示,LD活性也并无明显改变(P0.05)。RT-PCR、western blot结果显示:药物作用后HL-60细胞中ICAT表达水平上调(均P0.01);β-catenin表达水平下调(均P0.01);细胞核中β-catenin蛋白的水平下调(P0.01);TCF-4表达水平无明显改变(均P0.05);细胞核中的TCF-4无明显改变(P0.05);Wnt信号通路下游靶基因c-myc、cyclin D1、TCF-1的m RNA和蛋白表达水平均下调(均P0.05)。免疫荧光染色结果显示:细胞核中的β-catenin蛋白减少,TCF-4无明显改变。结论NSC67657可诱导白血病细胞HL-60向单核系分化,抑制细胞增殖,但不诱导细胞凋亡,且对细胞无毒副作用;在HL-60向单核系诱导分化的过程中,ICAT表达上调,Wnt信号通路关键调节因子β-catenin及下游靶基因的表达下调。提示:ICAT可能通过抑制Wnt/β-catenin信号通路参与了NSC67657诱导白血病细胞HL-60向单核系分化的过程。
[Abstract]:Objective to establish a cell model of HL-60 differentiation into monocytes induced by NSC6767, a new sterol drug, and to investigate the effect of NSC6767 on the biological behavior of leukemia cells. Methods HL-60 cells were treated with 10 渭 mol/L NSC67657 for 5 days to construct a cell differentiation model. HL-60 cells were treated with DMSO solvent for 5 days as control group. The differentiation direction of the cells was analyzed by using Rayleigh staining and alkaline 伪 -NBEase staining. Cell differentiation, cell cycle distribution and apoptosis were analyzed by flow cytometry. Lactate dehydrogenase (LDD) assay kit was used to detect the cytotoxicity of the drug to cells. RT-PCR and Western blot were used to analyze ICAT-catenin. The expression of m RNA and protein of T cell factor 4 (TCF-4) before and after differentiation; 尾 -catenin was detected by Western blot and immunofluorescence staining before and after differentiation. The downstream target gene c-myc of Wnt signaling pathway was analyzed by RT-PCR and Western blot. The expression of cyclin D _ (1) cyclin D _ (1) RNA and TCF-1 m RNA and protein before and after cell differentiation; Results the results showed that after 5 days of treatment with 10 渭 mol/L NSC67657, the cytoplasm increased significantly, the nucleoli disappeared, and the nucleus was round or irregular. The results of 伪 -NBE staining showed that the cytoplasm of the control group was colorless and negative. In the experimental group, bright red granules were found in the cytoplasm, which were positive and could be inhibited by sodium fluoride. The results of flow cytometry showed that the number of CD14 cells in the experimental group treated with 10 渭 mol/L NSC67657 for 5 days was 94.37 卤2.84). %. Higher than control group. [The percentage of G1 / G0 phase cells in the experimental group was 76.46 卤2.83%, which was higher than that in the control group. [The percentage of S phase cells in the experimental group was 18.76 卤0.98%, which was lower than that in the control group. [The difference was statistically significant (P 0.05). There were no significant changes in early and late apoptosis of HL-60 cells before and after drug treatment (all P 0.05). The results of LD test showed that the activity of LD did not change P0.05. RT-PCR. The results of western blot showed that the expression of ICAT in HL-60 cells was up-regulated (all P 0.01). The expression of 尾 -catenin was down-regulated (P 0.01). The level of 尾 -catenin protein in the nucleus was down-regulated (P 0.01). There was no significant change in the expression of TCF-4 (P 0.05). There was no significant change in TCF-4 in the nucleus. The downstream target gene of Wnt signaling pathway, c-myc cyclin D1, was detected. The expression of m RNA and protein in TCF-1 were all down-regulated (P0.05). The results of immunofluorescence staining showed that the 尾 -catenin protein in the nucleus decreased. Conclusion NSC67657 can induce the differentiation of leukemic cells into monocytes and inhibit the proliferation of leukemia cells, but it does not induce apoptosis and has no toxic and side effects on leukemic cells. The expression of HL-60 was up-regulated during its differentiation into monocytes. The expression of 尾 -catenin and downstream target gene in Wnt signaling pathway was down-regulated. ICAT may be involved in the differentiation of HL-60 into monocytes induced by NSC67657 by inhibiting Wnt- 尾 -catenin signaling pathway.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.7

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