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

【摘要】：目的：探討地西他濱(decitabine,DAC)對(duì)急性髓系白血病細(xì)胞株HL-60和急性淋系白血病細(xì)胞株Molt-4可能的作用和機(jī)制。 方法：用不同濃度DAC作用HL-60和Molt-4細(xì)胞24h、48h和72h,采用改良MTT法檢測(cè)HL-60和Molt-4細(xì)胞的增殖抑制作用；Annexin V-FITC/PI雙染法及PI單染法流式細(xì)胞儀檢測(cè)DAC對(duì)這兩種細(xì)胞凋亡及細(xì)胞周期的影響；RT-PCR檢測(cè)DAC對(duì)這兩種細(xì)胞P15、IER3、DNMT1、DNMT3A、DNMT3BmRNA表達(dá)作用的影響。 結(jié)果： (1)MTT結(jié)果顯示：DAC對(duì)HL-60和Molt-4細(xì)胞均有增殖抑制作用,并呈時(shí)間和劑量依賴性,且DAC對(duì)HL-60細(xì)胞的增殖抑制作用強(qiáng)于Molt-4細(xì)胞。24、48、72h, HL-60細(xì)胞的半數(shù)抑制濃度(IC50)分別為：5.78×104、44.86、0.614；Molt-4細(xì)胞分別為：3.07×106、6.30×103、3.06×102。 (2) Annexin V-FITC/PI雙染法檢測(cè)細(xì)胞凋亡結(jié)果顯示：隨著作用時(shí)間和藥物濃度的增加,DAC對(duì)HL-60和Molt-4細(xì)胞株均有促凋亡和壞死作用,尤以凋亡為主,且對(duì)HL-60細(xì)胞的促凋亡和壞死作用均強(qiáng)于Molt-4細(xì)胞。 (3)PI單染法流式細(xì)胞儀結(jié)果顯示：DAC作用后使HL-60細(xì)胞G2/M期的細(xì)胞比例增加,Molt-4細(xì)胞G0/G1期細(xì)胞比例減少,S期細(xì)胞比例增加,有時(shí)效和量效關(guān)系。 (4) RT-PCR結(jié)果顯示：HL-60和Molt-4細(xì)胞株P(guān)15、IER3mRNA的基礎(chǔ)表達(dá)水平均較低,DAC作用后,兩株細(xì)胞P15、IER3mRNA的表達(dá)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(diǎn)：(1)HL-60細(xì)胞在24h,5umol/L,P15、IER3mRNA的表達(dá)量最大,分別為1.377±0.095,1.113±0.002。(2)Molt-4細(xì)胞IER3mRNA的最大表達(dá)量則是出現(xiàn)在24h最大藥物濃度200umol/L作用下。 (5)DAC作用后,兩株細(xì)胞：(1) DNMT1mRNA的表達(dá)均減少；(2)DNMT3AmRNA的表達(dá)均無明顯影響；(3) DNMT3BmRNA的表達(dá)在HL-60細(xì)胞中減少,在Molt-4細(xì)胞中也呈一定在下降趨勢(shì)。 結(jié)論： (1)DAC呈時(shí)間和劑量依賴性抑制HL-60細(xì)胞和Molt-4細(xì)胞的生長(zhǎng),阻滯HL-60細(xì)胞于G2/M期,阻滯Molt-4細(xì)胞于S期,并促進(jìn)HL-60細(xì)胞和Molt-4細(xì)胞的凋亡,其對(duì)HL-60細(xì)胞的增殖抑制作用及促細(xì)胞凋亡作用均強(qiáng)于Molt-4細(xì)胞。 (2)在HL-60細(xì)胞和Molt-4細(xì)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(diào)P15和IER3基因的表達(dá)。 (3)IER3基因在血液系統(tǒng)惡性腫瘤中可能扮演一種抑癌基因的角色,隨著對(duì)IER3基因在惡性血液疾病中作用機(jī)制和表觀遺傳學(xué)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(diǎn)。 (4)DAC對(duì)髓系細(xì)胞株的作用強(qiáng)于淋系細(xì)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(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R96

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 王信峰;徐瑞容;楊力;姜?jiǎng)偃A;丁潤(rùn)生;陸偉;;地西他濱聯(lián)合全反式維甲酸對(duì)SKM-1 MDS細(xì)胞株的體外研究[J];交通醫(yī)學(xué);2009年02期

2 肖艷華;易紅;譚潭;梁婷;陳主初;肖志強(qiáng);;5-aza-2dC誘導(dǎo)白血病細(xì)胞分化及對(duì)Annexin A1/A2表達(dá)和甲基化狀態(tài)的影響[J];國(guó)際病理科學(xué)與臨床雜志;2008年03期

3 李小雨;沈建箴;沈松菲;付海英;周華蓉;吳淡森;張媛媛;鄭永青;;惡性血液病細(xì)胞株中IEX-1基因啟動(dòng)子區(qū)CpG島甲基化狀態(tài)的初探[J];中國(guó)實(shí)驗(yàn)血液學(xué)雜志;2011年02期

4 龍潺;唐雪元;廖巧芳;劉順妹;胡俊;;地西他濱聯(lián)合CAG方案在急性復(fù)發(fā)難治性急性髓系白血病中的初步應(yīng)用[J];中國(guó)實(shí)用醫(yī)藥;2013年19期

5 韓新愛;曾慧蘭;韓艷萍;孫爾維;;地西他濱對(duì)NB4及K562細(xì)胞增殖和凋亡的影響[J];中國(guó)實(shí)驗(yàn)血液學(xué)雜志;2013年02期

，

本文編號(hào)：1460239