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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 to investigate whether phenylhexylisothiocyanate can demethylate multiple myeloma U266 cell lines with hypermethylation of p16 gene. U266 cell lines were incubated with phenylhexylisothiocyanate (0,5,10μmol / L), and the DNA was extracted. Normal human genomic DNA was treated with bisulfite and used as a template for PCR amplification. A set of special fluorescently labeled probes was designed to construct a chip that detects the methylation changes at the three CpG sites in the promoter region of the p16 gene. The special fluorescently labeled probes include a pair of unmethylated probes and methylated probes , Detect the adjacent three CpG sites methylation level. Using gene chip method, a completely unmethylated DNA and fully methylated DNA were mixed to form a standard curve. The U266 cell sample was processed on chip and compared with the standard curve to obtain a quantitative detection result. The results showed that the repeatability and accuracy of the probe on the chip were very good. The methylation levels of p16 gene in U266 cells treated with 0,5,10μmol / L phenylhexyl isothiocyanate were 78.2%, 61.7 %, 54.8%. Conclusion: Phenylhexyl isothiocyanate can reduce the methylation of p16 gene in U266 cell line.