淋巴瘤Daudi细胞SHP-1甲基化及5-杂氮脱氧胞苷抑制性效应的研究  

SHP-1 gene's methylation status of Daudi lymphoma cell and the demethylation effect of 5-aza-2'-deoxycytidine

作　　者：方建晨[1] 苏祖兰[2] 邱耕 何丹[2] 冯智英[2] 朱明芬[4] 李莉[4] 何向蕾 

机构地区：[1]宁波市医疗中心 [2]中山大学第三附属医院病理科,广州510630 [3]中山大学基础医学院 [4]中山大学第三附属医院传染科实验室

出　　处：《中华血液学杂志》2006年第10期670-674,共5页Chinese Journal of Hematology

基　　金：国家自然科学基金资助项目（39870328）;广东省科技厅重点攻关项目（C30904）

摘　　要：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�Objective To explore the transcription regulation of 5-aza-2＇-deoxyeytidine （5-Aza-CdR） on SHP-1 gene and its effects on Daudi cell line growth. Methods MTF method and flow cytometry were used to detect the growth and apoptosis of Daudi cells after treated with different dosage of 5-Aza-CdR. Bisulrite sequencing PCR （BSP）, T-A cloning and sequence analysis were evaluated for methylation status. The SHP-1 mRNA and protein were determined by reverse transcription polymerase chain reaction （RT-PCR） ,immunohistochemistry. Results ①After 7 d treatment with 2.00 μ mol/L of 5-Aza-CdR, all cytosines （C） in Daudi cells genone DNA were converted to thymidine, and SHP-1 mRNA and protein expressed again in the cells while those Cs in CpG dinucleotides in untreated Daudi cells remained Cs; ②5-Aza-CdR inhibited the cell growth,The effects within certain extent were dose and time dependent：after 72 h treatrent with 5-Aza-CdR at 200. 00,20. 00,2. 00 and 0. 20 μ mol/L, the inhibitive rates were 72. 0% , 65. 1% , 51. 5% ,28.8% ,23.4% respectively;③5-Aza-CdR increased apoptosis rate of tumor cells with a dose and times dependent manner within certain extent, too ： at the 1,3,5 d treatment with 5-Aza-CdR 2.00 μmol/L, the apoptosis rates were 2.3% ,10.8% and 17.1% ; respectively. ④5-Aza-CdR also changed cell cycle of tumor cells： at 24 h treatmant with 5-Aza-CdR 2.00 μmol/L,92.7% tumor cells stopped at S phase and G1 phase cells were increased gradually with time. Conclusion DNA promoter hypermethylation is asscioated with SHP-1 gene silence in Daudi lymphoma cell line. 5-Aza-CdR could effectively cause demethylation and inhibit the growth of tumor cell by reactivating the gene transcription.

关 键 词：细胞系 淋巴瘤 抑制剂 脱氧胞苷 甲基化 蛋白质酪氨酸磷酸酶 基因表达 调控 细胞凋亡 

分 类 号：R733.1[医药卫生—肿瘤]