地西他滨对HL-60细胞株、Molt-4细胞株的作用和机制研究

发布时间：2018-01-24 14:14

本文关键词： 地西他滨(DAC) HL-60细胞株 MOLT-4细胞株抑癌基因 DNA甲基转移酶(DNMT)　出处：《昆明医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：探讨地西他滨(decitabine,DAC)对急性髓系白血病细胞株HL-60和急性淋系白血病细胞株Molt-4可能的作用和机制。方法：用不同浓度DAC作用HL-60和Molt-4细胞24h、48h和72h,采用改良MTT法检测HL-60和Molt-4细胞的增殖抑制作用；Annexin V-FITC/PI双染法及PI单染法流式细胞仪检测DAC对这两种细胞凋亡及细胞周期的影响；RT-PCR检测DAC对这两种细胞P15、IER3、DNMT1、DNMT3A、DNMT3BmRNA表达作用的影响。结果： (1)MTT结果显示：DAC对HL-60和Molt-4细胞均有增殖抑制作用,并呈时间和剂量依赖性,且DAC对HL-60细胞的增殖抑制作用强于Molt-4细胞。24、48、72h, HL-60细胞的半数抑制浓度(IC50)分别为：5.78×104、44.86、0.614；Molt-4细胞分别为：3.07×106、6.30×103、3.06×102。 (2) Annexin V-FITC/PI双染法检测细胞凋亡结果显示：随着作用时间和药物浓度的增加,DAC对HL-60和Molt-4细胞株均有促凋亡和坏死作用,尤以凋亡为主,且对HL-60细胞的促凋亡和坏死作用均强于Molt-4细胞。 (3)PI单染法流式细胞仪结果显示：DAC作用后使HL-60细胞G2/M期的细胞比例增加,Molt-4细胞G0/G1期细胞比例减少,S期细胞比例增加,有时效和量效关系。 (4) RT-PCR结果显示：HL-60和Molt-4细胞株P15、IER3mRNA的基础表达水平均较低,DAC作用后,两株细胞P15、IER3mRNA的表达随药物浓度的增加而增加,尤以作用24h增加幅度最大。但也各有特点：(1)HL-60细胞在24h,5umol/L,P15、IER3mRNA的表达量最大,分别为1.377±0.095,1.113±0.002。(2)Molt-4细胞IER3mRNA的最大表达量则是出现在24h最大药物浓度200umol/L作用下。 (5)DAC作用后,两株细胞：(1) DNMT1mRNA的表达均减少；(2)DNMT3AmRNA的表达均无明显影响；(3) DNMT3BmRNA的表达在HL-60细胞中减少,在Molt-4细胞中也呈一定在下降趋势。结论： (1)DAC呈时间和剂量依赖性抑制HL-60细胞和Molt-4细胞的生长,阻滞HL-60细胞于G2/M期,阻滞Molt-4细胞于S期,并促进HL-60细胞和Molt-4细胞的凋亡,其对HL-60细胞的增殖抑制作用及促细胞凋亡作用均强于Molt-4细胞。 (2)在HL-60细胞和Molt-4细胞中,DAC可能主要通过抑制DNMT1的活性,降低P15和IER3基因的甲基化水平,而上调P15和IER3基因的表达。 (3)IER3基因在血液系统恶性肿瘤中可能扮演一种抑癌基因的角色,随着对IER3基因在恶性血液疾病中作用机制和表观遗传学改变的深入研究,其可能成为恶性血液疾病靶向治疗的新靶点。 (4)DAC对髓系细胞株的作用强于淋系细胞株,这是否提示我们可将DAC优先用于髓系白血病的治疗和巩固治疗的临床探索和研究中。
[Abstract]:Aim: to investigate the possible effect and mechanism of decitabine DAC on acute myeloid leukemia cell line HL-60 and acute lymphoid leukemia cell line Molt-4. Methods: HL-60 and Molt-4 cells were treated with different concentrations of DAC for 48 h and 72 h. The proliferation inhibition of HL-60 and Molt-4 cells was detected by modified MTT assay. Annexin V-FITC / Pi double staining and Pi single staining flow cytometry were used to detect the effect of DAC on apoptosis and cell cycle. RT-PCR was used to detect the effect of DAC on the expression of DNMT3B mRNA in these two cell lines. Results: The results of HL-60 and Molt-4 showed that the proliferation of both HL-60 and Molt-4 cells was inhibited in a time-and dose-dependent manner. The inhibitory effect of DAC on the proliferation of HL-60 cells was stronger than that on Molt-4 cells for 72 hours. The half inhibitory concentration (IC50) of HL-60 cells was #number0# 5.78 脳 10 ~ (4) ~ (4) ~ (44.86) ~ (0.614); The number of Molt-4 cells was 3.07 脳 10 6, 6.30 脳 10 3 and 3.06 脳 10 2, respectively. The results of Annexin V-FITC / Pi double staining showed that with the increase of action time and drug concentration. DAC can promote apoptosis and necrosis of HL-60 and Molt-4 cell lines, especially on HL-60 cells, and it is stronger than Molt-4 cells in promoting apoptosis and necrosis of HL-60 cells. The results of flow cytometry showed that the proportion of G _ 2 / M phase of HL-60 cells increased and the proportion of G _ 0 / G _ 1 phase of Molt-4 cells decreased. The proportion of S phase cells increased and there was a time-dependent and dose-effect relationship. (4) RT-PCR results showed that the basic expression level of IER3 mRNA in the two cell lines P15 and HL-60 was lower than that in the Molt-4 cell line, and the P15 mRNA expression level in the two cell lines was lower than that in the control group. The expression of IER3mRNA increased with the increase of drug concentration, especially at 24 h. The expression of P15 IER3 mRNA was the highest (1.377 卤0.095). The maximum expression of IER3mRNA in Molt-4 cells was observed at the concentration of 200umol / L at 24 h. The expression of DNMT1mRNA in the two cell lines was decreased after treatment. The expression of DNMT3A mRNA was not significantly affected by the expression of DNMT3A mRNA. The expression of DNMT3BmRNA decreased in HL-60 cells and decreased in Molt-4 cells. Conclusion: It inhibited the growth of HL-60 cells and Molt-4 cells in a time-and dose-dependent manner, blocking HL-60 cells in G _ 2 / M phase and Molt-4 cells in S phase. It also promoted the apoptosis of HL-60 cells and Molt-4 cells. The inhibition of proliferation and apoptosis of HL-60 cells was stronger than that of Molt-4 cells. (2) in HL-60 cells and Molt-4 cells, the methylation level of P15 and IER3 genes may be reduced by inhibiting the activity of DNMT1. The expression of P15 and IER3 genes was up-regulated. IER3 gene may play a role as a tumor suppressor gene in hematological malignancies. With the further study of the mechanism and epigenetic changes of IER3 gene in malignant hematological diseases. It may become a new target for the treatment of malignant blood diseases. The effect of DAC on myeloid cell line is stronger than that on lymphocytic cell line, which suggests that DAC can be applied to the treatment of myeloid leukemia and the clinical research of consolidation therapy.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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