中文摘要：

目的检测骨髓增生异常综合征（MDS）细胞株MUTZ-1及原代细胞PTEN基因的甲基化情况，分析尿多酸肽（CDA-Ⅱ）对其影响及机制，探讨CDA-Ⅱ在MDS靶向治疗中的价值。方法采用Western blot法和RT．PCR检测MUTZ-1细胞及MDS患者原代细胞DNA甲基转移酶（DNMTs）和PTEN表达情况及CDA-Ⅱ对其的影响；采用甲基化特异性PCR（MSP）检测MUTZ-1细胞及MDS患者原代细胞PTEN甲基化情况及CDA-Ⅱ对其的影响。结果PTEN在高危MDS和MDS转化的急性髓系白血病（AML）患者细胞中完全甲基化；高危MDS和MDS转化的AML患者细胞高表达DNMTs，以DNMT3b最为明显。MUTZ-1细胞经2、4及8mg／mlCDA—Ⅱ处理后lYFEN蛋白表达量分别为0．23＋0．11、0．54±0．11及0．92±0．13，高于空白对照组的0．02±0．01（P〈0．01）；4mg／ml CDA-Ⅱ处理12、24及48h后PTEN蛋白表达量分别为0．15±0．06、0．52±0．12及0．89±0．13，高于空白对照组的0．02±0．01（P〈0．01）。CDA=Ⅱ明显下凋MUTZ-1细胞DNMTs（DNMTI、DNMT3a、DNMT3b）表达并导致其PTEN去甲基化，呈浓度依赖性（r=0．999、0．992、0．995，P〈值均0．01）和时问依赖性（r=0．989、0．981、0．985，P值均〈O．05），以对DNMTl作用最显著（F=21．256，P〈0．05）。结论PTEN基因在高危MDS患者中存在高度甲基化，靶向PTEN的去甲基化治疗可作为MDS治疗的新策略；CDA—Ⅱ通过抑制DNMTs导致PTEN去甲基化从而诱导MDS细胞凋亡。

英文摘要：

Objective To investigate the methylation patterns of PTEN gene in myelodysplastic syndrome （MDS） cell line MUTZ- 1 cells and the primary cells, to explore the effects of uroacitides （CDA-Ⅱ ） on the methylation patterns, and to provide novel target therapy for MDS. Methods Reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blot were used to determine the expression of PTEN and DNA methyltransferases （DNMTs） as well as the effects of CDA- Ⅱ on them. Methylation specific PCR （MSP） was used to identify the methylation patterns of PTEN and the effects of CDA- Ⅱ on them. Results PTEN was completely methylated in high-risk MDS and acute myeloid leukemia （AML） transformed from MDS. High expressions of DNMTs proteins, especially DNMT3b protein were found in high-risk MDS and AML cells transformed from MDS. PTEN expression level was increased from 0.02±0.01 at control group to 0.23±0.11, 0.54±0.11 and 0.92±0.13 after treatment with CDA- Ⅱ at 2, 4 and 8 mg/ml （P〈0,01）. If the cells were treated with CDA-Ⅱ at 4 mg/ml for 12, 24 and 48 hours, PTEN expression level was up-regulated to 0.15±0.06, 0.52±0.12 and 0.89±0.13, which were significantly higher than that of control group 0.02±0.01 （P〈0.01）. CDA-Ⅱ could significantly down- regulate the expression of DNMTs in MUTZ-1 cells in a dose- （r = 0.999, 0.992, 0.995, P〈0.01 ） and time- dependent （r=0.989, 0.981, 0.985, P〈0.05） manner, which led to the demethylation of PTEN. Among them, DNMT1 was the most down-regulated （F-21.256, P〈0.05）. Conclusion PTEN gene was hypermethylated in high-risk MDS, and target therapy of PTEN demethylation might be a new strategy for MDS therapy. CDA- Ⅱ could downreglate the expression of DNMTs and lead to PTEN demethylation which induce MUTZ-1 cells apoptosis.