慢性髓细胞白血病疾病分期与ZO-1基因甲基化关系研究

发布时间：2018-04-09 16:22

  本文选题：白血病　切入点：髓细胞　出处：《吉林大学》2015年硕士论文

【摘要】：背景：慢性髓细胞白血病（CML）简称慢粒，为恶性血液系统疾病，发生于骨髓造血干细胞，病程分为慢性期（CP）、加速期（AP）、最终急变期（BP/BC）。加速期、急变期被认为是疾病进展期，本病中位发病年龄在60岁~65岁。目前酪氨酸激酶抑制剂（TKI）成为慢粒治疗的一线用药，传统观点认为慢粒患者进入急性期首选异基因造血干细胞移植，且为可能治愈本病的唯一手段。否则病程仅为3~6个月，但移植费用昂贵且存在风险。所以临床中治疗以维持患者处于慢性期不进展为佳，需重点关注慢粒患者疾病何时发生进展，但目前并没有明确的分子标志物提示慢粒急变。研究显示肿瘤的发生发展同表观遗传学密切相关，其中DNA甲基化是表观遗传调控的重要机制，甲基化异常是白血病发生过程中的一个普遍现象。研究表明在各种白血病中发现了多个基因高甲基化状态，提示DNA甲基化异常修饰同白血病发病相关。前期研究工作显示，人紧密连接蛋白（ZO-1）基因启动子区，在急性白血病中呈现特异性高甲基化状态，，与急性白血病发生、发展及转归密切相关，是白血病预后一个不良因素。 目的：对慢粒和急性白血病的细胞系以及慢粒患者病程不同时期的骨髓标本，进行ZO-1基因启动子区甲基化状态检测，探讨慢粒疾病不同时期ZO-1基因启动子区甲基化状态的差异，及其与疾病分期转变之间的关系。 方法：应用甲基化特异性聚合酶链反应（MS-PCR）及亚硫酸氢钠联合限制性内切酶分析法（COBRA）检测K562细胞及HL-60、Molt4等细胞，ZO-1基因启动子区甲基化状态，ZO-1基因的表达情况，以及去甲基化药物对ZO-1基因甲基化状态及表达的影响。检测CML不同疾病分期骨髓标本ZO-1甲基化状态，并通过扩大临床标本数目，探讨不同分期CML患者中ZO-1基因甲基化阳性患者比例。并对一例CML患者在不同分期阶段骨髓标本检测，检测其慢性期、急变期、急变期治疗后回到慢性期时ZO-1基因启动子区甲基化状态及表达的变化。 结果： 1. ZO-1基因启动子区在K562细胞系中呈现非甲基化状态，在HL-60、Molt-4细胞系中呈现特异性高甲基化状态，同时在急性白血病细胞系中ZO-1表达沉默。经去甲基化药物地西他滨处理后的HL-60、Molt-4细胞系，ZO-1基因启动子区由高甲基化状态转化为非甲基化状态，同时恢复ZO-1基因的表达。 2. ZO-1基因启动子区在正常人群呈现非甲基化状态，慢粒患者慢性期呈现非甲基化状态，慢粒患者加速期及急变期中呈现特异性高甲基化状态。对10例CML-CP患者检测，ZO-1基因均为非甲基化状态，对8例CML-AP患者检测，4例（50%）呈现高甲基化状态，CML-BC患者7例，5例（70%）呈现ZO-1基因高甲基化状态。 3.针对1例慢粒患者在慢性期呈现ZO-1基因非甲基化，急变期呈现高甲基化状态，恢复至慢性期后甲基化状态可转变回非甲基化状态。 结论： ZO-1基因启动子区甲基化状态同慢粒疾病不同时期有相关性。随着疾病进展，ZO-1基因甲基化比例升高，出现高甲基化状态提示可能有疾病进展倾向。
[Abstract]:Background: chronic myeloid leukemia (CML) CML, malignant hematological diseases, bone marrow hematopoietic stem cells, the course is divided into chronic phase (CP), accelerated phase (AP), the final blast phase (BP/BC). The acceleration period is considered in blastic phase progression of the disease, the disease incidence in at the age of 60 years. At present, ~65 tyrosine kinase inhibitors (TKI) become the first-line treatment of CML, the traditional view is that in acute phase of CML patients preferred allogeneic hematopoietic stem cell transplantation, and is the only possible means to cure the disease. It is only 3~ duration of 6 months, but the cost is high and there is a risk of transplant. So the clinical treatment of patients in chronic period not to maintain progress is good, need to focus on disease progression when CML patients, but there is no clear molecular marker for leukemia. Research shows that the occurrence and development of tumors with epigenetic density Closely related, in which DNA methylation is an important epigenetic regulation mechanism of table, abnormal methylation of leukemia is a common phenomenon in the process. The results show that multiple gene hypermethylation was found in all kinds of leukemia, suggesting that DNA methylation modification with leukemia disease. Previous study showed that people the tight junction protein (ZO-1) gene promoter region showed high specificity of methylation status in acute leukemia, the occurrence and development of acute leukemia, and the prognosis is closely related to the prognosis of leukemia is a bad factor.
Objective: to chronic and acute leukemia cell lines and the course of disease in patients with CML bone marrow specimens of different periods, promoter methylation status of ZO-1 gene promoter of detection, difference of CML in different periods of ZO-1 gene promoter methylation status, and the relationship between the change and staging of the disease.
Methods: methylation specific polymerase chain reaction (MS-PCR) and Sodium Bisulfite joint restriction enzyme analysis (COBRA) detection of K562 cells and HL-60 Molt4 cells, ZO-1 gene promoter methylation and expression of ZO-1 gene, and the effects of demethylation drugs on the expression and methylation status of ZO-1 gene detection of CML in different stages of disease. Bone marrow specimens of ZO-1 methylation, and by expanding the number of clinical specimens, explore the different stages of ZO-1 patients with CML gene methylation in patients with positive proportion. And on a case of CML patients in different stages of bone marrow specimens detection, detection of the chronic phase, acute phase, acute phase after treatment to chronic period the ZO-1 gene promoter and expression of promoter methylation status.
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