中文摘要：

目的 研究甲基化抑制剂5-杂氮脱氧胞苷（5-aza-2’deoxycytidine，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-1 mRNA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G1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，期细胞数量随培养时间延长而增加，药物作用1，3，5d时，G1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

英文摘要：

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.