巢式MSP检测砷剂诱导人多发性骨髓瘤U266细胞系p16基因去甲基化及转录.doc

巢式 MSP 检测砷剂诱导人多发性骨髓瘤 U266
细胞系 p16 基因去甲基化及转录

【摘要】 本研究探讨用高灵敏度的 DNA 甲基化检测方法及 DNA

克隆测序分析法检测三氧化二砷（As2O3）的去甲基化作用，并对其可

能的去甲基化作用机制进行分析。 采用巢式甲基特异性 PCR 法 nested （

methylation specific PCR， n MSP） DNA 克隆测序分析法检测 、

As2O3 作用前后 U266 细胞株 p16 基因甲基化状态，应用 RT PCR 检

测 p16、DNA 甲基转移酶 1（DNMT1） DNMT3A、DNMT3B 基因 mRNA 的表 、

达，以生长曲线、MTT 法、集落形成实验检测 As2O3 对骨髓瘤细胞生

长和增殖的抑制作用。 利用流式细胞仪 DNA 含量分析法探讨 As2O3 对

多发性骨髓瘤细胞系 U266 周期的影响。结果表明： ①未处理组 U266

细胞基因组 DNA 的胞嘧啶保持不变,而经 As2O3 作用的 U266 细胞基因

组 DNA 的胞嘧啶均已变为胸腺嘧啶，这说明 U266 细胞存在 p16 基因

甲基化， As2O3 作用后 p16 基因异常甲基化的现象被逆转； ②未处

理组细胞 p16 基因不表达，As2O3 作用 72 小时后 p16 基因表达增强，

0.5 mmol/L 组、1.0 mmol/组和 2.0 mmol/组 p16 基因表达阳性条

带灰度值与b 肌动蛋白比值分别为(0.22±0.10)、(0.59±0.11)、

(0.68±0.09)，阳性对照灰度比值为(0.77±0.13)，差异有非常显著

的统计学意义（P<0.01） ；③与未处理组相比，As2O3 作用 72 小时

后甲基转移酶（DNMT）1、DNMT3A、DNMT3B 的表达下降并呈浓度依赖

性；④与对照组相比，3 组不同浓度 As2O3 均能明显抑制骨髓瘤细胞

生长，G0-G1 期细胞增加。结论： As2O3 可能通过抑制甲基转移酶

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（DNMT1） DNMT3A、DNMT3B 和（或）直接对 p16 基因去甲基化，使 、

p16 基因表达上调，恢复其活性，从而实现其对细胞周期的调控功能，

将细胞阻滞于 G0-G1 期，抑制骨髓瘤细胞的增长。

【关键词】 巢式 MSP p16 基因去甲基化 三氧化二砷 多发性骨髓

瘤 U266 细胞 甲基转移酶

n MSP Detection of p16 Gene Demethylation and

Transcription in Human Multiple Myeloma U266 Cell Line Induced

by Arsenic Trioxide

Abstract The study was purposed to investigate the effect

of arsenic trioxide(As2O3) induced p16 gene demethylation by

a sensitive and specific PCR based method (nested

methylation specific PCR, n MSP) and DNA sequencing for rapid

analysis of the promoter demethylation status, and to explore

the possible mechanism of the p16 gene demethylation in human

multiple myeloma U266 cells induced by As2O3. The methylation

status of the p16 gene in U266 cell line before and after

treatment with As2O3 was detected by the nested methylation

specific PCR and DNA sequencing, the mRNA of p16, DNA

methyltransferase (DNMT 1、DNMT3A and 3B) gene were determined

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by RT PCR, and the induced growth inhibition of U266 cell was

assayed by growth curve, MTT and CFU; the DNA content of U266

cells was analyzed by flow cytometry after being exposed to As2O3.

The results showed that (1) all cytosines in CpG dinucleotides

in untreated U266 cell not were changed , while all cytosines

in treated U266 cells with As2O3 had been converted to thymidine.

(2) p16 gene was not expressed in U266 cell line after

methylation. As compared with the b actin, the expression of

U266 cell p16 gene mRNA was increased to ( 0.22±0.10)、(0.59

±0.11)、(0.68±0.09) after exposed to 0.5 mmol/L、1.0 mmol/L

and 2.0 mmol/L As2O3 for 72 hours respectively . (3) As2O3 could

significantly down regulate DNA methyltransferase 1(DNMT 1)、

DNMT3A and DNMT3B gene at mRNA level in a dose dependent manner.

(4) U266 cells line grew slowly and arrested at G0-G1 phase after

treatment with three different concentrations of As2O3. It is

concluded that As2O3 can activate and up regulate the

expression of p16 gene which inhibits the proliferation of U266

cell through inducing the G0-G1 arrest by demethylation or/and

by inhibiting DNMT 1、DNMT3A and 3B gene.

近年来的研究显示， 的异常甲基化是一种与肿瘤发生密切相 DNA

关的表遗传学机制, 其启动子区 Cp G 岛的异常甲基化已成为抑癌基

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