异硫氰酸苯己酯诱导Molt-4细胞p15基因去甲基化的实验研究

Inhibition of gene p15 hypermethylation by phenylhexyl isothiocyanate in Molt-4 cells

目的研究异硫氰酸苯己酯（PHI）对急性T淋巴细胞性白血病Molt-4细胞p15基因的去甲基化作用及转录激活作用。方法采用甲基特异性聚合酶链反应（MSP）检测PHI作用前后Molt-4细胞株p15基因甲基化状态的变化情况；RT-PCR检测p15基因的mRNA的表达变化；Western blotting检测p15蛋白的表达变化。结果PHI作用于Molt-4细胞5d后，p15基因的异常甲基化现象被逆转，基因的甲基化程度减弱；基因转录激活，p15 mRNA、p15蛋白表达呈浓度依赖性增加。各组p15 mRNA条带灰度值与β-actin比值为：空白对照组（0．17±0．12），PHI 10μmol／L组（0．29±0．14），PHI 20μmol／L组（0．55±0．07），PHI 40μmol／L组（0．93±0．13），各加药组与空白对照组相比，差异均有统计学意义（P〈0．05）。结论PHI有DNA去甲基化的作用，能诱导沉默的p15基因重新表达。

Objective To investigate the effect of phenylhexyle isothiocyanate （PHI） demethylation and activation of transcription gene p15 in acute leukemia cell line Molt-4. Methods DNA sequencing and modified methylation specific PCR （MSP） were used to screen p15-M and p15-U mRNA after Molt-4 cells were treated with PHI. p15 mRNA was measured by RT-PCR. P15 protein was detected by Western blotting. Results Hypermethylation of gene p15 was apparently attenuated and activation of transcription p15 gene was de novo after 5 days exposure to PHI. PHI enhanced both the expression of p15 mRNA and p15 protein in a concentration-dependent manner. The ratio of the gray scale of p15 mRNA strap was 0.17±0.12 in control, 0.29±0.14 in PHI 10 μmol/L, 0.55±0.07 in PHI 20 μmol/L, 0.93±0.13 in PHI 40 μmol/L. Conclusion PHI could active demethylation and transcription of gene p15.