地西他濱、三氧化二砷對(duì)HL-60細(xì)胞端粒酶活性影響的實(shí)驗(yàn)研究
[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.
【學(xué)位授予單位】:河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】:碩士
【學(xué)位授予年份】:2017
【分類號(hào)】:R733.71
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