二烯丙基二硫作用于HL-60细胞后凋亡相关基因的差异表达  

Effects of Diallyl Disulfide on Differential Expression of Apoptosis-associated Genes in Leukemia Cell Line HL-60

作　　者：林敏[1] 谢海龙[1] 苏琦[1] 周建国[1] 谭晖[1] 易岚[1] 唐海林[1] 黄炎[1] 石莺[1] 

机构地区：[1]南华大学肿瘤研究所,湖南衡阳421001

出　　处：《癌症》2007年第4期351-356,共6页Chinese Journal of Cancer

基　　金：湖南省教育厅科研基金优秀青年项目(No.04B057);湖南省教育厅科研项目(No.03C391;No.06C696)~~

摘　　要：背景与目的:二烯丙基二硫(diallyl disulfide,DADS)对多种肿瘤细胞有促凋亡作用,白血病是儿童最常见的恶性肿瘤,近期研究提示DADS能够在体外诱导人白血病细胞发生凋亡,但其具体作用机制尚不清楚。本实验旨在研究DADS诱导人白血病细胞HL-60凋亡的生物学效应,并探讨其分子机制。方法:运用DNA含量分析、AnnexinⅤ/PI双标记流式细胞仪、DNA琼脂糖凝胶电泳以及透射电子显微镜形态学观察等方法来证实细胞凋亡。通过基因芯片检测60μmol/L DADS作用于HL-60细胞4h后凋亡相关基因的表达谱,采用RT-PCR技术验证上调基因Fas-L和下调基因Bag-1。结果:15、30、60和120μmol/L DADS作用于HL-60细胞24h后,DNA含量分析出现了明显的亚二倍体峰;60μmol/LDADS作用4、8、12、24h后,Annexin Ⅴ/PI双标流式细胞仪检测表明早期凋亡细胞显著增加。60μmol/LDADS作用24h后,在DNA琼脂糖凝胶上可见特征性的梯形条带。电子显微镜观察到细胞体积缩小,核浓缩和凋亡小体形成等典型的形态学改变。通过基因芯片检测发现,60μmol/L DADS作用4h后有8个凋亡相关基因表达差异显著,选择其中Fas-L和Bag-1两个基因运用RT-PCR技术进行验证,其结果与基因芯片结果一致。结论:DADS能够诱导人白血病细胞HL-60凋亡,这可能是多个基因和多条信号转导通路共同作用的结果。BACKGROUND ＆ OBJECTIVE： Diallyl disulfide （DADS） can induce apoptosis in various cancer cell lines in vitro. Leukemia is the most common pediatric malignancy. Recent studies have suggested that DADS can induce apoptosis in human leukemia cells, but its mechanisms remain unclear. This study was to investigate the biological effects of DADS on the apoptosis of human leukemia cell line HL-60, and explore its molecular mechanisms. METHODS： After treatment of DADS, cell apoptosis was verified by flow cytometry with Annexin V/PI staining, DNA agarose gel electrophoresis, transmission electron microscopy. The expression profile of apoptosis-associated genes in HL-60 cells, with or without 4-hour treatment of DADS （60 μmol/L）, was identified by gene array. The up-regulated Fas-L gene and down-regulated Bag-1 transcription-polymerase chain reaction gene were confirmed by reverse （RT-PCR）. RESULTS： When treated with 15, 30, 60, 120 μmol/L DADS for 24 h, HL-60 cells presented obvious subdiploid peaks. When treated with 60 μmol/L DADS for 4, 8, 12, 24 h, early apoptotic cells were greatly increased. When treated with 60 μmol/L DADS for 24 h, DNA extracted from HL-60 cells displayed a characteristic ladder pattern on agarose gel electrophoresis; typical morphologic apoptotic changes were observed under electron microscope, including cell shrinkage, nuclear condensation, and formation of apoptotic bodies; the differential expression of 8 apoptosis-associated genes were found with gene array. The expression of Fas-L and Bag-1 detected by RT-PCR were consistent with those detected by gene array. CONCLUSION： DADS could induce apoptosis in HL-60 cells, which might be mediated by some specific genes and various signal transduction pathways.

关 键 词：二烯丙基二硫/药理学 白血病 HL-60细胞 凋亡 基因芯片 

分 类 号：R733.7[医药卫生—肿瘤]