地西他滨、三氧化二砷对HL-60细胞端粒酶活性影响的实验研究
发布时间：2018-08-27 16:52
【摘要】：目的:急性髓系白血病(acute myeloid leukemia,AML)是一类造血干细胞恶性克隆性疾病,是严重威胁人类健康的疾病。AML主要的治疗方法为化疗,近年来成人AML患者经标准“3+7”化疗方案治疗后,完全缓解率在70%-80%。同时由于微小残留病灶(MRD)的存在,缓解后复发是AML病人的危险因素。只有近20%-30%的AML病人可以获得无病生存[1]。异基因造血干细胞移植是治疗AML最有效的方法,由于患者客观条件限制较多(如供着来源、经济状况)等多方面的因素,目前能够接受造血干细胞移植的AML病人为少数。因此寻找新的靶向药物提高化疗药物的效果是我们不断探索的领域。急性髓系白血病的发生与细胞遗传学、分子遗传学改变有密切关系,研究发现大多数急性髓系白血病(AML)患者中存在2种或2种以上基因异常甲基化,DNA甲基化在白血病中广泛存在,启动子Cp G岛超甲基化导致的抑癌基因失活与白血病的发生和肿瘤细胞化疗药物抵抗密切相关[2]。地西他滨(decitabine,DAC),又称为5-氮杂-2’-脱氧胞苷,是一种胞嘧啶核苷类似物,为特异的DNA甲基化转移酶抑制剂,是目前研究较多的甲基化抑制剂,可通过逆转Cp G岛甲基化,使多种因甲基化而失活的抑癌基因重新表达,抑制肿瘤细胞生长而达到治疗肿瘤的目的[3]。三氧化二砷(arsenic trioxide,AS2O3)是传统中药砒霜的有效成分之一,是治疗急性早幼粒细胞白血病的经典药物之一,该药物通过诱导白血病细胞分化及凋亡两条途径发挥其治疗作用,随着对该药物的不断研究,现在逐渐应用于其他类型的恶性血液病的治疗中[4]。目前去甲基化药物不断应用于临床,不仅应用于骨髓增生异常综合征的患者,同时针对老年AML、复发、难治的AML患者,有一定的效果。研究新的靶向药物,以提高药物疗效并延长无病生存期是目前研究的重要课题。本实验研究DAC、AS2O3对HL-60细胞(人急性髓系白血病细胞株)增殖、凋亡的作用以及对HL-60细胞端粒酶的基因及蛋白表达水平变化的的影响,探讨去甲基化药物对AML的作用机制,旨在为今后临床用药提供更多的理论依据。方法:选取HL-60(人急性髓系白血病)细胞株为研究对象,实验随机分组:设置未加药物空白对照组,单药DAC组(1μmol/L、10μmol/L、50μmol/L),单药AS2O3组(1.25μmol/L、2.5μmol/L、5μmol/L)、DAC联合AS2O3组(10μmol/L+2.5μmol/L、10μmol/L+5μmol/L、50μmol/L+2.5μmol/L、50μmol/L+5μmol/L),DAC(50μmol/L)预处理+AS2O3组(1.25μmol/L、2.5μmol/L、5μmol/L)。根据不同浓药物度作用HL-60细胞后,检测不同时间增殖、凋亡的作用以及对端粒酶基因及蛋白表达水平的影响。每组实验重复3次。1用CCK8法筛查药物浓度,检测不同药物浓度作用HL-60细胞24h、48h、72h后的增殖抑制率;2用流式细胞术(FCM)检测不同药物浓度作用HL-60细胞24h、48h、72h后的凋亡率;3用RT-PCR法检测不同药物浓度作用HL-60细胞48h后端粒酶的h TERT m RNA表达水平的变化;4用Western Blot法检测不同药物浓度作用HL-60细胞48h后h TERT的蛋白表达水平的变化;5用SPSS 21.0统计软件进行统计学处理。所有数据用3次独立实验的平均值表示,实验结果均采用均数±标准差(X±S)表示;两组比较应用t检验,多组比较应用方差分析,P0.05有统计学意义。结果:1 DAC、AS2O3单药对HL-60细胞株增殖抑制率的检测。1.1不同浓度DAC、AS2O3单药作用于HL-60细胞24h、48h、72h后,单药DAC、AS2O3增加药物浓度和作用时间延长,可增加增殖抑制率。单药DAC作用HL-60细胞后,增殖抑制率由(10.85±2.04)%增加到(56.01±2.04)%,单药AS2O3作用HL-60细胞后,增殖抑制率由(14.55±1.68)%增加到(85.09±3.67)%。各药物处理组之间比较差异有统计学意义(P=0.00)。说明DAC、AS2O3单药均对HL-60细胞有抑制增殖的作用,并呈时间-剂量依赖性。(图1,图2,表1,表2)1.2 DAC联合AS2O3对HL-60细胞作用24h、48h、72h后,不同时间段两药联合作用HL-60细胞,增加药物联合浓度,其增殖抑制率由(35.68±1.98)%增加到(94.89±1.02)%,各联合处理组与单药处理组比较增殖抑制率明显增高,比较有统计学意义(P=0.00)。说明DAC联合AS2O3对HL-60细胞的抑制作用优于单药。(图3,表3)1.3经DAC(50μmol/L)预处理后,不同浓度AS2O3对HL-60细胞作用24h、48h、72h后,增加药物浓度和延长作用时间,可增强增殖抑制率,增殖抑制率由(26.98±0.67)%增加到(93.10±0.80)%,与AS2O3单药组比较增殖抑制率显著增加,各药物处理组间及与AS2O3单药组之间比较差异具有统计学意义(P=0.00)。说明DAC预处理可以增强AS2O3对HL-60细胞的增殖抑制作用。(图4,表4)2 DAC、AS2O3单药对HL-60细胞株凋亡的影响2.1单药DAC、AS2O3作用于HL-60细胞24h、48h、72h后,凋亡率逐渐升高。单药DAC对HL-60细胞凋亡率由(7.61±1.02)%增加到(36.33±0.51)%,单药AS2O3对HL-60细胞凋亡率由(9.80±0.26)%增加到(40.56±0.90)%。各药物处理组间以及与空白对照组之间比较差异有统计学意义(P=0.00)。说明DAC、AS2O3单药能够诱导HL-60细胞凋亡,并呈时间-剂量依赖性。(图5,表5,表6)2.2 DAC联合AS2O3对HL-60细胞作用24h、48h、72h后,凋亡率由(18.00±1.37)%增加到(57.76±0.61)%,各联合组与单药组比较凋亡率显著增加,且差异具有统计学意义(P=0.00)。说明DAC联合AS2O3对HL-60细胞诱导凋亡具有协同作用。(图5,表7)2.3经DAC(50μmol/L)预处理后,不同浓度的AS2O3对HL-60细胞作用24h、48h、72h后,凋亡率随药物浓度的增加和作用时间的延长而增加,凋亡率由(17.93±0.55)%增加到(60.96±0.70)%,较AS2O3单药组凋亡率明显增加。各AS2O3浓度经DAC预处理后与AS2O3单药组比较差异有统计学意义(P=0.00)。说明DAC预处理可以增强AS2O3对HL-60细胞诱导凋亡的作用。(图5,表8)3 DAC、AS2O3对HL-60细胞内端粒酶h TERT m RNA表达水平变化的影响3.1不同浓度DAC、AS2O3单药对HL-60细胞作用48h后,增加药物浓度,端粒酶h TERT m RNA表达水平有下降。单药DAC(0μmol/L、1μmol/L、10μmol/L、50μmol/L),对h TERT m RNA的表达量由0.993±0.007下降到0.578±0.004;单药AS2O3(0μmol/L、1.25μmol/L、2.5μmol/L、5μmol/L)对h TERT m RNA的表达量由0.993±0.007下降到0.657±0.034。各处理组间h TERT m RNA比较,差异具有统计学意义(P=0.00)。说明DAC、AS2O3单药能够降低HL-60细胞内端粒酶h TERT m RNA基因的表达水平,呈剂量依赖性。(图6,表9,表10)3.2不同浓度的DAC联合AS2O3对HL-60细胞作用48h后,增加联合组药物浓度,对h TERT m RNA的表达量由0.528±0.022下降到0.213±0.023。各处理组之间h TERT m RNA表达水平差异具有统计学意义(P=0.00)。说明DAC联合AS2O3能够增加HL-60细胞内端粒酶h TERT m RNA的表达水平,且两者具有协同作用。(图6,表11)3.3经DAC(50μmol/L)预处理后,不同药物浓度的AS2O3对HL-60细胞作用48h后,增加药物浓度,h TERT m RNA的表达量由0.161±0.022下降到0.065±0.008,较AS2O3单药组比较表达水平显著下降,h TERT m RNA各处理组间及与单药AS2O3比较h TERT m RNA差异均具有统计学意义(P=0.00)。说明DAC预处理可以增加AS2O3对HL-60细胞内端粒酶h TERT m RNA的抑制作用。(图6,表12)4 DAC、AS2O3对HL-60细胞端粒酶内h TERT蛋白表达水平变化的影响4.1不同浓度DAC、AS2O3单药对HL-60细胞作用48h后,随着药物浓度的增加,端粒酶h TERT的蛋白表达水平有下降。单药DAC(0μmol/L、1μmol/L、10μmol/L、50μmol/L)对h TERT的蛋白相对表达量由1.046±0.073下降到0.576±0.063;单药AS2O3(0μmol/L、1.25μmol/L、2.5μmol/L、5μmol/L)对h TERT的蛋白相对表达量由1.046±0.073下降到0.426±0.014。各处理组间及处理组与空白对照组之间比较差异具有统计学意义(P=0.00)。说明DAC、AS2O3单药能够降低HL-60细胞内端粒酶h TERT蛋白的表达,并呈剂量依赖性。(图7,表13,表14)4.2不同浓度的DAC联合AS2O3对HL-60细胞作用48h后,随着药物浓度的增加,对h TERT的蛋白相对表达量由0.674±0.016下降到0.111±0.015,且差异具有统计学意义(P=0.00)。说明DAC联合AS2O3能够抑制HL-60细胞端粒酶h TERT蛋白的表达,且两者具有协同作用。(图8,表15)4.3经DAC(50μmol/L)预处理后,不同药物浓度的AS2O3对HL-60细胞作用48h后,增加药物浓度,端粒酶的h TERT的蛋白相对表达量由0.311±0.015下降到0.055±0.011,较AS2O3单药组蛋白相对表达量显著下降,各药物处理组间及各处理组与AS2O3单药组间比较差异具有统计学意义(P=0.00)。说明DAC预处理可以提升AS2O3对HL-60细胞端粒酶h TERT蛋白的抑制作用。(图9,表16)结论:1不同浓度地西他滨和三氧化二砷及两药联合作用HL-60细胞株后,均有抑制增殖、诱导凋亡的作用,并呈剂量-时间依赖性。2地西他滨和三氧化二砷发挥作用具有协同效应。3经地西他滨预处理后,可以增加三氧化二砷对HL-60细胞株的敏感性,其作用机制可能与降低端粒酶h TERT的表达有关,端粒酶h TERT可能是DAC诱导HL-60细胞凋亡的调控基因之一。
[Abstract]:Objective: Acute myeloid leukemia (AML) is a malignant clonal disease of hematopoietic stem cells and a serious threat to human health. Chemotherapy is the main treatment for AML. In recent years, the complete remission rate of adult AML patients is 70% - 80% after the standard "3 + 7" chemotherapy regimen. Alien hematopoietic stem cell transplantation is the most effective method to treat AML. Due to the limited objective conditions (such as source of supply, economic status) and other factors, it is now possible to accept hematopoietic stem cell transplantation. The incidence of acute myeloid leukemia is closely related to cytogenetics and molecular genetics. It has been found that there are two or more abnormal gene methylation, DN in most patients with acute myeloid leukemia (AML). A-methylation is widespread in leukemia, and the inactivation of tumor suppressor genes caused by hypermethylation of promoter Cp G islands is closely related to the occurrence of leukemia and the resistance of tumor cells to chemotherapeutic drugs [2].Decitabine (DAC), also known as 5-aza-2'-deoxycytidine, is a cytosine nucleoside analogue that inhibits specific DNA methylation transferase Preparations, as methylation inhibitors, have been extensively studied. They can reverse the methylation of Cp G islands, re-express many tumor suppressor genes inactivated by methylation, inhibit the growth of tumor cells and achieve the purpose of tumor treatment [3]. Arsenic trioxide (AS2O3) is one of the effective components of traditional Chinese medicine arsenic, and is an early treatment for acute diseases. Meiocytic leukemia is one of the classical drugs. It plays a therapeutic role by inducing differentiation and apoptosis of leukemic cells. With the continuous study of this drug, it is gradually used in the treatment of other types of malignant hematological diseases [4]. It is an important subject to study new targeted drugs to improve the efficacy of drugs and prolong disease-free survival. In this study, the effects of DAC and AS2O3 on proliferation, apoptosis and apoptosis of HL-60 cells (human acute myeloid leukemia cell line) were studied. To investigate the effect of demethylation drugs on the expression of telomerase gene and protein in HL-60 cells, and to explore the mechanism of demethylation drugs on AML, so as to provide more theoretical basis for clinical use in the future. DAC group (1 micromolmolmol/L, 10 micromolmol/L, 10 micromol/L, 50 micromolmolmolmolmol/L, 50 micromolmolmolmol/L), single drug AS2O3 group (1.25 micromolmol/L, 2.5 micromolmol/L, 5 micromolmolmolmol/L, 5 micromolmol/L), DAC combined with AS2O3 group (10 micromolmol/L+2.5 micromol/L, 10 micromolmolmolmolmol/L, 10 micromolmolmol/L/L, 10 10 micromolmolmolmolmolmolmolmolmolmol/L/L, 10 10 micromolmolmolmolmolmolmolmolmolmolL/L/L/L 10 10 10 0/L, 10 micromolmolmolmolmolmolmolmolmolmolmolmolmolL/L/L 10 10 0/L 10(2) After HL-60 cells were treated with different concentrations of the drug, The proliferation and apoptosis of HL-60 cells at different time and the expression of telomerase gene and protein were detected. The apoptotic rate of HL-60 cells was measured by RT-PCR. The expression of H TERT m RNA was detected by RT-PCR. The expression of H TERT m RNA was detected by Western Blot. The expression of H TERT protein was analyzed by SPSS 21.0 software. Results: 1 DAC, AS2O3 single drug inhibited the proliferation of HL-60 cells. 1.1 Different concentrations of DAC, AS2O3 single drug acted on HL-60 cells 24 hours, 48 hours, 72 hours, single drug DAC, A The inhibitory rate of proliferation of HL-60 cells treated with single drug DAC increased from (10.85 [2.04]% to (56.01 [2.04])%. The inhibitory rate of proliferation of HL-60 cells treated with single drug AS2O3 increased from (14.55 [1.68]% to (85.09 [3.67]%). There was a significant difference between the treatment groups (P The results showed that DAC and AS2O3 could inhibit the proliferation of HL-60 cells in a time-and dose-dependent manner. (Fig. 1, 2, 1, 2) 1.2 DAC combined with AS2O3 could inhibit the proliferation of HL-60 cells for 24, 48 and 72 hours, and the inhibitory rate increased from (35.68 (1.98)% to (94.89 (1.02)) The inhibitory effect of DAC combined with AS2O3 on HL-60 cells was superior to that of single drug (P = 0.00). (Fig. 3, Table 3) After pretreatment with DAC (50 micromol/L), AS2O3 at different concentrations increased the inhibitory effect on HL-60 cells for 24 hours, 48 hours and 72 hours. The inhibition rate of proliferation increased from (26.98 (Fig. 4, Table 4) 2 DAC, AS2O3 monotherapy on apoptosis of HL-60 cell line 2.1 single drug DAC, AS2O3 on HL-60 cell 24 hours, 48 hours, 72 hours, the apoptosis rate gradually increased. Single drug DAC on HL-60 cell apoptosis rate increased from (7.61 [1.02]% to (36.33 [0.51)%]%, single drug AS2O3 on HL-60 cell apoptosis rate increased from (9.80 [0.26]% to (40.56 [0.90)%]. The results showed that DAC and AS2O3 could induce apoptosis of HL-60 cells in a time-and dose-dependent manner. (Fig. 5, Table 5, Table 6) 2.2 DAC combined with AS2O3 could induce apoptosis of HL-60 cells 24 hours, 48 hours and 72 hours later, the apoptosis rate increased from (18.00 (1.37)% to (57.76 (0.61)% in each combination group compared with the single drug group. The apoptosis rate of HL-60 cells was significantly higher than that of HL-60 cells, and the difference was statistically significant (P=0.00). It showed that DAC combined with AS2O3 had synergistic effect on HL-60 cells. (Fig. 5, Table 7) After pretreatment with DAC (50 micromol/L), the apoptosis rate of HL-60 cells treated with different concentrations of AS2O3 for 24 hours, 48 hours, 72 hours increased with the increase of drug concentration and prolonged action time, and withered. The apoptosis rate of HL-60 cells increased from (17.93 6550 Effects of different concentrations of DAC, AS2O3 monotherapy on HL-60 cells 48 hours after treatment, increased drug concentration, telomerase h TERT m RNA expression level decreased. Single drug DAC (0 micromol/L, 1 micromol/L, 10 micromol/L, 50 micromol/L), the expression of H TERT m RNA decreased from 0.993 (+0.007) to 0.578 (+0.004); single drug AS2O3 (0 micromol/L, 1.25 micromol/L, 2.25 micromol/L). The expression of H TERT m RNA in HL-60 cells was decreased from 0.993.007 to 0.657.034 by 5 The expression of H TERT m RNA in HL-60 cells decreased from 0.528 (+0.022) to 0.213 (+0.023) 48 hours after the combination of DAC and AS2O3. There was a significant difference in the expression level of H TERT m RNA between the treatment groups (P = 0.00). It indicated that DAC combined with AS2O3 could increase the expression level of H TERT m RNA in HL-60 cells, and the expression level of H TERT m RNA in both groups was increased. After pretreatment with DAC (50 micromol/L), the expression of H TERT-m RNA decreased from 0.161 (+ 0.022) to 0.065 (+ 0.008) after 48 hours of treatment with different concentrations of AS2O3, which was significantly lower than that of AS2O3 alone. The expression of H TERT-m RNA was significantly lower in each treatment group and compared with that of AS2O3 alone. The difference of TERT m RNA was statistically significant (P = 0.00). It indicated that DAC pretreatment could increase the inhibitory effect of AS2O3 on telomerase h TERT m RNA in HL-60 cells. (Fig. 6, Table 12) 4 DAC, AS2O3 on the expression of H TERT protein in HL-60 cells 4. The relative expression of H TERT protein decreased from 1.046.073 to 0.576.063 in DAC (065 The results showed that DAC and AS2O3 could decrease the expression of telomerase h TERT protein in HL-60 cells in a dose-dependent manner. (Fig. 7, Table 13, Table 14)4.2 Different concentrations of DAC combined with AS2O3 could increase the concentration of DAC in HL-60 cells 48 hours after treatment. The relative expression of H TERT protein decreased from 0.674 [0.016] to 0.111 [0.015], and the difference was statistically significant (P = 0.00). It indicated that DAC combined with AS2O3 could inhibit the expression of H TERT protein in HL-60 cells, and the two had synergistic effects. (Fig. 8, Table 15) After pretreatment with DAC (50 um mol/L), AS2O3 at different concentrations acted on HL-60 cells. After 48 hours of treatment, the relative expression of H TERT protein of telomerase decreased from 0.311 (+ 0.015) to 0.055 (+ 0.011), which was significantly lower than that of AS2O3. There was a significant difference between the treatment groups and the AS2O3 group (P = 0.00). Inhibitory effect of H TERT protein on telomerase activity in HL-60 cells. (Fig. 9, Table 16) Conclusion: 1 Different concentrations of dicitabine, arsenic trioxide and their combination can inhibit proliferation and induce apoptosis of HL-60 cells in a dose-time dependent manner. After treatment, arsenic trioxide can increase the sensitivity of HL-60 cells. The mechanism may be related to the decrease of telomerase h TERT expression. Telomerase h TERT may be one of the regulatory genes of DAC-induced apoptosis of HL-60 cells.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.71
本文编号：2207868
[Abstract]:Objective: Acute myeloid leukemia (AML) is a malignant clonal disease of hematopoietic stem cells and a serious threat to human health. Chemotherapy is the main treatment for AML. In recent years, the complete remission rate of adult AML patients is 70% - 80% after the standard "3 + 7" chemotherapy regimen. Alien hematopoietic stem cell transplantation is the most effective method to treat AML. Due to the limited objective conditions (such as source of supply, economic status) and other factors, it is now possible to accept hematopoietic stem cell transplantation. The incidence of acute myeloid leukemia is closely related to cytogenetics and molecular genetics. It has been found that there are two or more abnormal gene methylation, DN in most patients with acute myeloid leukemia (AML). A-methylation is widespread in leukemia, and the inactivation of tumor suppressor genes caused by hypermethylation of promoter Cp G islands is closely related to the occurrence of leukemia and the resistance of tumor cells to chemotherapeutic drugs [2].Decitabine (DAC), also known as 5-aza-2'-deoxycytidine, is a cytosine nucleoside analogue that inhibits specific DNA methylation transferase Preparations, as methylation inhibitors, have been extensively studied. They can reverse the methylation of Cp G islands, re-express many tumor suppressor genes inactivated by methylation, inhibit the growth of tumor cells and achieve the purpose of tumor treatment [3]. Arsenic trioxide (AS2O3) is one of the effective components of traditional Chinese medicine arsenic, and is an early treatment for acute diseases. Meiocytic leukemia is one of the classical drugs. It plays a therapeutic role by inducing differentiation and apoptosis of leukemic cells. With the continuous study of this drug, it is gradually used in the treatment of other types of malignant hematological diseases [4]. It is an important subject to study new targeted drugs to improve the efficacy of drugs and prolong disease-free survival. In this study, the effects of DAC and AS2O3 on proliferation, apoptosis and apoptosis of HL-60 cells (human acute myeloid leukemia cell line) were studied. To investigate the effect of demethylation drugs on the expression of telomerase gene and protein in HL-60 cells, and to explore the mechanism of demethylation drugs on AML, so as to provide more theoretical basis for clinical use in the future. DAC group (1 micromolmolmol/L, 10 micromolmol/L, 10 micromol/L, 50 micromolmolmolmolmol/L, 50 micromolmolmolmol/L), single drug AS2O3 group (1.25 micromolmol/L, 2.5 micromolmol/L, 5 micromolmolmolmol/L, 5 micromolmol/L), DAC combined with AS2O3 group (10 micromolmol/L+2.5 micromol/L, 10 micromolmolmolmolmol/L, 10 micromolmolmol/L/L, 10 10 micromolmolmolmolmolmolmolmolmolmol/L/L, 10 10 micromolmolmolmolmolmolmolmolmolmolL/L/L/L 10 10 10 0/L, 10 micromolmolmolmolmolmolmolmolmolmolmolmolmolL/L/L 10 10 0/L 10(2) After HL-60 cells were treated with different concentrations of the drug, The proliferation and apoptosis of HL-60 cells at different time and the expression of telomerase gene and protein were detected. The apoptotic rate of HL-60 cells was measured by RT-PCR. The expression of H TERT m RNA was detected by RT-PCR. The expression of H TERT m RNA was detected by Western Blot. The expression of H TERT protein was analyzed by SPSS 21.0 software. Results: 1 DAC, AS2O3 single drug inhibited the proliferation of HL-60 cells. 1.1 Different concentrations of DAC, AS2O3 single drug acted on HL-60 cells 24 hours, 48 hours, 72 hours, single drug DAC, A The inhibitory rate of proliferation of HL-60 cells treated with single drug DAC increased from (10.85 [2.04]% to (56.01 [2.04])%. The inhibitory rate of proliferation of HL-60 cells treated with single drug AS2O3 increased from (14.55 [1.68]% to (85.09 [3.67]%). There was a significant difference between the treatment groups (P The results showed that DAC and AS2O3 could inhibit the proliferation of HL-60 cells in a time-and dose-dependent manner. (Fig. 1, 2, 1, 2) 1.2 DAC combined with AS2O3 could inhibit the proliferation of HL-60 cells for 24, 48 and 72 hours, and the inhibitory rate increased from (35.68 (1.98)% to (94.89 (1.02)) The inhibitory effect of DAC combined with AS2O3 on HL-60 cells was superior to that of single drug (P = 0.00). (Fig. 3, Table 3) After pretreatment with DAC (50 micromol/L), AS2O3 at different concentrations increased the inhibitory effect on HL-60 cells for 24 hours, 48 hours and 72 hours. The inhibition rate of proliferation increased from (26.98 (Fig. 4, Table 4) 2 DAC, AS2O3 monotherapy on apoptosis of HL-60 cell line 2.1 single drug DAC, AS2O3 on HL-60 cell 24 hours, 48 hours, 72 hours, the apoptosis rate gradually increased. Single drug DAC on HL-60 cell apoptosis rate increased from (7.61 [1.02]% to (36.33 [0.51)%]%, single drug AS2O3 on HL-60 cell apoptosis rate increased from (9.80 [0.26]% to (40.56 [0.90)%]. The results showed that DAC and AS2O3 could induce apoptosis of HL-60 cells in a time-and dose-dependent manner. (Fig. 5, Table 5, Table 6) 2.2 DAC combined with AS2O3 could induce apoptosis of HL-60 cells 24 hours, 48 hours and 72 hours later, the apoptosis rate increased from (18.00 (1.37)% to (57.76 (0.61)% in each combination group compared with the single drug group. The apoptosis rate of HL-60 cells was significantly higher than that of HL-60 cells, and the difference was statistically significant (P=0.00). It showed that DAC combined with AS2O3 had synergistic effect on HL-60 cells. (Fig. 5, Table 7) After pretreatment with DAC (50 micromol/L), the apoptosis rate of HL-60 cells treated with different concentrations of AS2O3 for 24 hours, 48 hours, 72 hours increased with the increase of drug concentration and prolonged action time, and withered. The apoptosis rate of HL-60 cells increased from (17.93 6550 Effects of different concentrations of DAC, AS2O3 monotherapy on HL-60 cells 48 hours after treatment, increased drug concentration, telomerase h TERT m RNA expression level decreased. Single drug DAC (0 micromol/L, 1 micromol/L, 10 micromol/L, 50 micromol/L), the expression of H TERT m RNA decreased from 0.993 (+0.007) to 0.578 (+0.004); single drug AS2O3 (0 micromol/L, 1.25 micromol/L, 2.25 micromol/L). The expression of H TERT m RNA in HL-60 cells was decreased from 0.993.007 to 0.657.034 by 5 The expression of H TERT m RNA in HL-60 cells decreased from 0.528 (+0.022) to 0.213 (+0.023) 48 hours after the combination of DAC and AS2O3. There was a significant difference in the expression level of H TERT m RNA between the treatment groups (P = 0.00). It indicated that DAC combined with AS2O3 could increase the expression level of H TERT m RNA in HL-60 cells, and the expression level of H TERT m RNA in both groups was increased. After pretreatment with DAC (50 micromol/L), the expression of H TERT-m RNA decreased from 0.161 (+ 0.022) to 0.065 (+ 0.008) after 48 hours of treatment with different concentrations of AS2O3, which was significantly lower than that of AS2O3 alone. The expression of H TERT-m RNA was significantly lower in each treatment group and compared with that of AS2O3 alone. The difference of TERT m RNA was statistically significant (P = 0.00). It indicated that DAC pretreatment could increase the inhibitory effect of AS2O3 on telomerase h TERT m RNA in HL-60 cells. (Fig. 6, Table 12) 4 DAC, AS2O3 on the expression of H TERT protein in HL-60 cells 4. The relative expression of H TERT protein decreased from 1.046.073 to 0.576.063 in DAC (065 The results showed that DAC and AS2O3 could decrease the expression of telomerase h TERT protein in HL-60 cells in a dose-dependent manner. (Fig. 7, Table 13, Table 14)4.2 Different concentrations of DAC combined with AS2O3 could increase the concentration of DAC in HL-60 cells 48 hours after treatment. The relative expression of H TERT protein decreased from 0.674 [0.016] to 0.111 [0.015], and the difference was statistically significant (P = 0.00). It indicated that DAC combined with AS2O3 could inhibit the expression of H TERT protein in HL-60 cells, and the two had synergistic effects. (Fig. 8, Table 15) After pretreatment with DAC (50 um mol/L), AS2O3 at different concentrations acted on HL-60 cells. After 48 hours of treatment, the relative expression of H TERT protein of telomerase decreased from 0.311 (+ 0.015) to 0.055 (+ 0.011), which was significantly lower than that of AS2O3. There was a significant difference between the treatment groups and the AS2O3 group (P = 0.00). Inhibitory effect of H TERT protein on telomerase activity in HL-60 cells. (Fig. 9, Table 16) Conclusion: 1 Different concentrations of dicitabine, arsenic trioxide and their combination can inhibit proliferation and induce apoptosis of HL-60 cells in a dose-time dependent manner. After treatment, arsenic trioxide can increase the sensitivity of HL-60 cells. The mechanism may be related to the decrease of telomerase h TERT expression. Telomerase h TERT may be one of the regulatory genes of DAC-induced apoptosis of HL-60 cells.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.71
【参考文献】
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