异硫氰酸苯己酯降低U266细胞株p16基因甲基化的研究  

Phenylhexyl Isothiocyanate Reducing U266 Cell Line Methylation Level of p16 Gene

作　　者：陈宝安[1] 寿倍明[1] 周冬蕊[2] 丁家华[1] 高冲[1] 孙耘玉[1] 王骏[1] 程坚[1] 赵刚[1] 宋慧慧[1] 鲍文[1] 刘德龙[3] 马旭东[4] 陆祖宏[2] 

机构地区：[1]东南大学临床医学院、东南大学附属中大医院血液科,江苏南京210009 [2]东南大学生物医学工程系芯片实验室、吴健雄实验室、国家重点实验室,江苏南京210096 [3]美国纽约医学院医学部,美国纽约10595 [4]福建省漳州市医院血液科,福建漳州350004

出　　处：《中国实验血液学杂志》2008年第5期1060-1063,共4页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目(编号39970832);国家自然科学基金项目(编号30600152)

摘　　要：本研究探讨异硫氰酸苯己酯对p16基因高甲基化的多发性骨髓瘤U266细胞株是否有去甲基化的作用。用异硫氰酸苯己酯0、5、10μmol/L孵育U266细胞株,提取DNA备用。用亚硫酸氢盐处理正常人基因组DNA,并以此为模板进行PCR扩增。设计1组特殊荧光标记探针以构建1种检测p16基因启动子区3个CpG位点甲基化改变的芯片,特殊荧光标记探针包括1对非甲基化探针和甲基化探针,检测相邻的3个CpG位点甲基化的程度。采用基因芯片的方法,将完全未甲基化的DNA和完全甲基化的DNA混合后制成标准曲线,将U266细胞株样本处理后点在芯片上与标准曲线进行比较后得出定量检测的结果。结果表明:芯片上探针的可重复性和精确性很好,0、5、10μmol/L异硫氰酸苯己酯处理后的U266细胞株p16基因的甲基化程度分别为78.2%、61.7%、54.8%。结论:异硫氰酸苯己酯可以降低U266细胞株p16基因甲基化的程度。This study was purposed to investigate whether phenylhexyl isothiocyanate （PHI） can reduce p16 gene methylation level or not. The myeloma U226 cell line was cultued with PHI of 0,5,10 μmol/L for 72 hours, then DNA was extracted. Hydrosulfite was used to treat the genome DNA of healthy adult, PCR amplification was carded out by using this DNA as template. The gene chip detecting rnethylation changes of 3 CpG in promoter region of p16 gene was constructed by designing a pair of probes which contain one methylated and one unmethylated probes. This pair of probes was used to detect 3 consecutive sites of CpG island in p16 gene. The standard curve was constructed by using gene chip after the methylated and unmethylated DNA were mixed at different ratio. Then treated samples of U266 cells were dotted on gene chip, obtained results were compared with standard curve to get the quantitative results. The results indicated that the probes on chip had excellent reproducible ability and precision, the methylation level of p16 gene in U266 cells treated with 0,5 and 10 μmol/L of PHI was determined as 78.2%, 61.7% and 54.8%, respectively. It is concluded that the PHI can reduce the methylation level of p16 gene in U266 cells.

关 键 词：异硫氰酸苯己酯 U266细胞株 P16基因甲基化 基因芯片 

分 类 号：R733.3[医药卫生—肿瘤]