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目的研究甲基化抑制剂5-杂氮脱氧胞苷(5-aza-2′-deoxyeytidine.5-Aza-CdR)对 Bur-kitt’s淋巴瘤细胞系 Daudi 细胞中的 SHP-1抑癌基因的转录调控作用及对 Daudi 细胞生长增殖的生物学影响,寻找肿瘤治疗新靶点:方法应用 MTT 法检测5-杂氮脱氧胞苷200.00,20.00,2.00,0.20μmol/L 等不同剂量,作用24,48,72h 对 Daudi 细胞存活率的影响。应用流式细胞术观察5-杂氮脱氧胞苷作用1,3,5d 细胞周期及凋亡率的变化。用亚硫酸氢盐测序 PCR(bisulfite sequencing PCR.BSP)、T-A 克隆及 DNA 测序分析甲基化状态。RT-PCR、免疫组织化学法检测5-杂氮脱氧胞苷(2.00μmol/L)处理前和处理7dDaudi 细胞 SHP-1 mRNA 及蛋白表达的变化。结果①经5-杂氮脱氧胞苷2.00μmol/L 作用7d 的 Daudi 细胞基因组 DNA 的胞嘧啶均已变为胸腺嘧啶,未经5-杂氮脱氧胞苷作用的对照组 Daudi 细胞基因组 DNA 的胞嘧啶保持不变;②经5-杂氮脱氧胞苷作用7d,SHP-1mRNA 和蛋白均重新表达;③5-杂氮脱氧胞苷抑制 Daudi 细胞的增殖,在一定范围内与药物浓度及作用时间呈正相关,药物作用72h,剂量为200.00,20.00,2.00和0.20μmol/L 时,瘤细胞增殖的抑制率分别为72.0%.65.1%.51.5%.28.8%和23.4%:④5-杂氮脱氧胞苷可使 Dandi 细胞凋亡率增加,且作用也呈时间依赖性,用药1,3,5d 细胞凋亡率分别为2.3%,10.8%和17.1%:⑤5-杂氮脱氧胞苷对于细胞周期的影响,主要体现在 S 期和 G_1期,最显著的是 5-杂氮脱氧胞苷2.00μmol/L 作用24h 后92.7%的细胞阻滞在 S 期;其次,在相同药物浓度下.G_1期细胞数量随培养时间延长而增加,药物作用1,3,5d 时,G_1期细胞分别占细胞总数的1.2%,7.9%和21.5%。结论 DNA 异常甲基化是导致 Daudi 细胞 SHP-1基因缄默的重要原因:特异性甲基转移酶抑制剂5-杂氮脱氧胞苷能较好地逆转 Daudi 细胞 DNA 异常甲基化,并有效地激活因高甲基化所致 SHP-1基因缄默的再转录,诱导该基因的表达,从而抑制肿瘤细胞生长。5-杂氮脱氧胞苷有望成为新型抗肿廇药物,对 Burkitts 淋巴瘤的疗效可能更佳。
Objective To investigate the transcription of SHP-1 tumor suppressor gene in Bur-kitt’s lymphoma cell line Daudi by methylation inhibitor 5-aza-2’-deoxyeytidine.5-Aza-CdR And the biological effects on the growth and proliferation of Daudi cells in order to find a new target for tumor therapy.Methods MTT assay was used to detect the effects of different doses of 5-aza-deoxycytidine 200.00,20.00,2.00,0.20μmol / , 72h on the survival rate of Daudi cells. Flow Cytometry was used to observe the changes of cell cycle and apoptosis rate after 1,3,5diazacytidine treatment. Methylation status was analyzed using bisulfite sequencing PCR. BSP, T-A cloning and DNA sequencing. RT-PCR and immunohistochemistry were used to detect the expression of SHP-1 mRNA and protein in DuDaudi cells treated with 5-aza-deoxycytidine (2.00μmol / L). Results ① Cytosine of genomic DNA of Daudi cells that had been treated with 5-aza-deoxycytidine (2.00μmol / L) for 7 days had been changed to thymine. Cells from the control group of Daudi cells without 5-aza-deoxycytidine Pyrimidine remained unchanged; ② SHP-1 mRNA and protein were re-expressed after 5-aza-deoxycytidine treatment for 7 days; ③ 5-aza-deoxycytidine inhibited the proliferation of Daudi cells within a certain range with drug concentration and duration The relative inhibition rates of proliferation of tumor cells were 72.0%, 65.1%, 51.5%, 28.8% and 23.4% at 72h and 200.00, 20.00, 2.00 and 0.20μmol / Dandi cells can increase the apoptosis rate, and the role of time-dependent manner, the 1,3,5 d apoptosis rates were 2.3%, 10.8% and 17.1%: ⑤ 5-aza-deoxycytidine on the cell cycle , Mainly in S phase and G_1 phase, most notably 92.7% cell arrest in S phase after treated with 2.00μmol / L 5-aza-deoxycytidine; secondly, in the same drug concentration, the number of cells in G_1 phase With the increase of culture time, G_1 phase cells accounted for 1.2%, 7.9% and 2% of the total number of cells respectively 1.5%. Conclusion Abnormal methylation of DNA is an important reason for the silencing of SHP-1 gene in Daudi cells. The specific methyltransferase inhibitor 5-aza-deoxycytidine can reverse DNA abnormal methylation in Daudi cells effectively and effectively Activation of silenced SHP-1 transcription due to hypermethylation induces the expression of this gene and thus inhibits tumor cell growth. 5-aza-deoxycytidine is expected to become a new anti-inflammatory drugs, the efficacy of Burkitts lymphoma may be better.