【作者】 王利芳；

【导师】 李登举；

【作者基本信息】 华中科技大学 ， 内科学， 2011， 硕士

【摘要】 【目的】研究5-氮杂-2′-脱氧胞苷(5-Aza-CdR)对人慢性粒细胞白血病(CML)急红变细胞系K562细胞生物学活性和DNA结合抑制因子4(ID4)基因表达的影响,探寻白血病基因治疗的新靶点。【方法】1.应用甲基化特异性PCR(MS-PCR)方法检测CML急红变细胞系K562细胞中ID4基因甲基化情况。2.实时荧光定量PCR(RQ-PCR)检测不同浓度5-Aza-CdR(0.5,2,5,10μM)处理K562细胞48h后ID4 mRNA的表达水平。3. FITC标记的膜联蛋白V/碘化丙锭(Annexin V-FITC/PI)双染色流式细胞术分析不同浓度5-Aza-CdR作用24h,48h,72h后细胞凋亡率的变化,并分析5-Aza-CdR作用48h后细胞周期的变化。【结果】K562细胞中存在ID4基因的甲基化;5-Aza-CdR处理后K562细胞ID4 mRNA表达增加,并具有浓度依赖性。ID4基因的表达水平在不同药物处理组之间差异具有统计学意义(P < 0.01)。5-Aza-CdR可使K562细胞凋亡率增加,并且作用呈时间剂量依赖性。5μM 5-Aza-CdR处理K562细胞24h,48h,72h后细胞凋亡率分别为15.3%,17.6%,21.3%。不同浓度5-Aza-CdR(0.5,2,5,10μM)处理K562细胞48h后细胞凋亡率分别为6.9%,12.6%,17.6%,29.3%。与对照组相比,统计学差异具有显著性(P<0.05)。5-Aza-CdR处理K562细胞48h后,随着药物浓度的增加,G0/G1期细胞逐渐增多,G2/M期细胞逐渐减少,细胞阻滞在G0/G1期。0μM、2μM和5μM 5-Aza-CdR处理48h后G0/G1期细胞分别为26.45%,33.49%,40.68%;G2/M期细胞分别为17.54%,8.44%,6.48%。处理组与对照组相比,均有显著统计学差异(P<0.01)。【结论】甲基化转移酶抑制剂5-Aza-CdR能促使CML急红变细胞系K562细胞中沉默的ID4基因重新表达,进而可能参与了K562细胞凋亡和细胞周期阻滞的调控。更多还原

【Abstract】 Objective: To study the effect of 5-Aza-CdR on the biological activity of human erythromyeloblastoid leukemia cell line K562 and the expression of inhibitor of DNA binding 4 (ID4), and to search the new target of gene therapy.Methods: 1. To detect the situation of ID4 methylation in K562 cell line by methylation specific PCR (MS-PCR). 2. To analyze the expression levels of ID4 mRNA in K562 cell line with the treatment of different concentrations of 5-Aza-CdR (0.5, 2, 5, 10μM) after 48h by RQ-PCR. 3. After treated by different concentrations of 5-Aza-CdR,K562 cells were stained with FITC labeled annexin V/propidium iodide (Annexin V-FITC/PI). The change of apoptosis rate and cell cycle of were analyzed by Flow Cytometry.Results: The K562 cells exist ID4 gene methylation. After 5-Aza-CdR is added to the medium, there is a dose dependent upregulation of the gene ID4 mRNA expression. The differences of the gene ID4 mRNA level among experimental groups are obvious (P < 0.01). 5-Aza-CdR can also induce the apoptosis of K562. The apoptosis rates of k562 cells are of time and dose dependent. 5μM 5-Aza-CdR results in 15.3%, 17.6% and 21.3% K562 cells apoptosis at 24h, 48h and 72h respectively. The apoptosis rates of K562 cells are 6.9%, 12.6%, 17.6% and 29.3% respectively after treated with different concentrations of 5-Aza-CdR (0.5, 2, 5, 10μM) for 48 hours. Compared with the control group (0μM), there is a significant difference (P <0.05). The change of cell cycle of K562 cells also are of dose dependent. Treatment of K562 cells with 5-Aza-CdR (2, 5μM) for 48h caused a 1.27-fold and 1.54-fold increase in K562 cells that arrest in G0/G1 phase in comparison with control cells. The treatment also caused a decease K562 cells that entered in G2/M phase. The treated K562 cells in G2/M phase were 0.48-fold and 0.37-fold of control group correspondingly. There is a significant statistical difference between treated groups and control group (P <0.01).Conclusions: Methyltransferase inhibitor 5-Aza-CdR can result in the re-expressions of the silent ID4 gene in K562 cells. The upregulation of ID4 maybe is key factor which is related with cell apoptosis and cell-cycle arrest of K562 cells treated by 5-Aza-CdR.更多还原