dads诱导hl60细胞g2m期阻滞差异表达基因的分离与克隆word格式论文.docxVIP

测序、比较同源性，发现 7 个新的 EST 片段，已经在 GenBank 中登录。其中 WT-1047仅在 DADS 处理人白血病 HL-60 细胞中有表达；采用 3?RACE 克隆出 WT-1047 的 3?端一段序列。结论：构建了 DADS 诱导人白血病 HL-60 细胞 G2/M 期阻滞差异表达 cDNA 文库， 该文库含有 120 个正向阳性克隆和 100 个反向阳性克隆；获得了 7 个新的 EST；克 隆了一条与 DADS 诱导人白血病 HL-60 细胞 G2/M 期阻滞新的相关基因。关键词：二烯丙基二硫、抑制消减杂交、差异表达、G2/M 期阻滞相关基因 RACE、 生物信息学、EST本研究受以下课题资助：1. 湖南省自然科学基金（03JJY3082）2. 湖南省自然科学基金重点项目(04 JJY2006)The isolation of differentially expressed genes in the G2/M arrest induced by diallyl disulfide in HL-60 cells using suppression subtractive hybridizationObjective: Diallyl disulfide(DADS), an oil-soluble allyl sulfur compound found in processed garlic, was found to inhibit the growth of various tumors by decreasing the cell growth rate, inducing apoptosis and cell cycle arrest. The previous studies in our laboratoryshowed that DADS induced the growth arrest of HL-60 cells in the G2/M phase，but thedetail mechanism by which DADS induced the cell cycle arrest remains to be elucidated. The aim of this study was to isolate and identify G2/M arrest-related genes, which were differentially expressed in HL-60 treated with or without DADS using suppression subtractive hybridization (SSH).Methods: An SSH cDNA library of HL-60 cells was constructed using DADS-treated HL-60 cell mRNA as tester and untreated-HL-60 cell mRNA as driver, respectively. Positive clones in the library were randomly selected. The patterns of the differentially expressing-gene fragments were analyzed by PCR. Sequences of resulting cDNA fragments were compared with that of genes in GenBank by Blastn.The resulting expressed sequence tags (EST) were confirmed by RT-PCR based on mRNA from HL-60 cells with or without DADS treatment. Both 3?and 5?-terminal sequences of one novel EST were amplified by rapid amplification of cDNA ends (RACE). The primers for RACE were designed in the basis of a highly conserved region in the mentioned EST by Primer5.0. The cDNA from 3?RACE was cloned into pGEM-T Easy Vector, and the positive clones were sequenced. The