本文關鍵詞： 地西他濱(DAC) HL-60細胞株 MOLT-4細胞株 抑癌基因 DNA甲基轉移酶(DNMT)　出處：《昆明醫(yī)科大學》2014年碩士論文　論文類型：學位論文

【摘要】：目的：探討地西他濱(decitabine,DAC)對急性髓系白血病細胞株HL-60和急性淋系白血病細胞株Molt-4可能的作用和機制。 方法：用不同濃度DAC作用HL-60和Molt-4細胞24h、48h和72h,采用改良MTT法檢測HL-60和Molt-4細胞的增殖抑制作用；Annexin V-FITC/PI雙染法及PI單染法流式細胞儀檢測DAC對這兩種細胞凋亡及細胞周期的影響；RT-PCR檢測DAC對這兩種細胞P15、IER3、DNMT1、DNMT3A、DNMT3BmRNA表達作用的影響。 結果： (1)MTT結果顯示：DAC對HL-60和Molt-4細胞均有增殖抑制作用,并呈時間和劑量依賴性,且DAC對HL-60細胞的增殖抑制作用強于Molt-4細胞。24、48、72h, HL-60細胞的半數抑制濃度(IC50)分別為：5.78×104、44.86、0.614；Molt-4細胞分別為：3.07×106、6.30×103、3.06×102。 (2) Annexin V-FITC/PI雙染法檢測細胞凋亡結果顯示：隨著作用時間和藥物濃度的增加,DAC對HL-60和Molt-4細胞株均有促凋亡和壞死作用,尤以凋亡為主,且對HL-60細胞的促凋亡和壞死作用均強于Molt-4細胞。 (3)PI單染法流式細胞儀結果顯示：DAC作用后使HL-60細胞G2/M期的細胞比例增加,Molt-4細胞G0/G1期細胞比例減少,S期細胞比例增加,有時效和量效關系。 (4) RT-PCR結果顯示：HL-60和Molt-4細胞株P15、IER3mRNA的基礎表達水平均較低,DAC作用后,兩株細胞P15、IER3mRNA的表達隨藥物濃度的增加而增加,尤以作用24h增加幅度最大。但也各有特點：(1)HL-60細胞在24h,5umol/L,P15、IER3mRNA的表達量最大,分別為1.377±0.095,1.113±0.002。(2)Molt-4細胞IER3mRNA的最大表達量則是出現在24h最大藥物濃度200umol/L作用下。 (5)DAC作用后,兩株細胞：(1) DNMT1mRNA的表達均減少；(2)DNMT3AmRNA的表達均無明顯影響；(3) DNMT3BmRNA的表達在HL-60細胞中減少,在Molt-4細胞中也呈一定在下降趨勢。 結論： (1)DAC呈時間和劑量依賴性抑制HL-60細胞和Molt-4細胞的生長,阻滯HL-60細胞于G2/M期,阻滯Molt-4細胞于S期,并促進HL-60細胞和Molt-4細胞的凋亡,其對HL-60細胞的增殖抑制作用及促細胞凋亡作用均強于Molt-4細胞。 (2)在HL-60細胞和Molt-4細胞中,DAC可能主要通過抑制DNMT1的活性,降低P15和IER3基因的甲基化水平,而上調P15和IER3基因的表達。 (3)IER3基因在血液系統惡性腫瘤中可能扮演一種抑癌基因的角色,隨著對IER3基因在惡性血液疾病中作用機制和表觀遺傳學改變的深入研究,其可能成為惡性血液疾病靶向治療的新靶點。 (4)DAC對髓系細胞株的作用強于淋系細胞株,這是否提示我們可將DAC優(yōu)先用于髓系白血病的治療和鞏固治療的臨床探索和研究中。
[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 鹵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.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R96

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