检测NPM基因突变并探讨NPM对白血病细胞生物学特性的影响

项目名称： 检测NPM基因突变并探讨NPM对白血病细胞生物学特性的影响

项目编号： No.30872418

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 张伶

作者单位： 重庆医科大学

项目金额： 30万元

中文摘要： 最近国内外文献报道近三分之一的急性髓系白血病患者可发生核仁磷酸蛋白（NPM）基因突变及NPM蛋白胞浆移位，提出一种新的白血病亚群（NPMc+ AML）。它的出现有助于完善白血病的WHO分型、预后判断和微量残留病监测，因此迫切需要建立适合临床的NPM突变检测方法，并进一步阐明NPM突变在白血病发病中的作用机制。本项目首先采用PCR技术在分子水平上检测NPM基因突变，同时利用荧光标记自制的突变型NPM蛋白单抗在细胞水平上检测胞浆蛋白；然后通过转染将携NPM突变基因或者NPM基因RNA干扰片段的病毒重组体导入白血病细胞系中，采用软琼脂克隆形成实验、FCM、RT-PCR和滤膜小室法等方法检测靶细胞增殖潜能、周期分布、凋亡特性和趋化性的改变，观察NPM突变对白血病细胞生物学特性的影响。本研究将推动NPM突变这项新指标在临床血液学检验的广泛应用，同时也有助于了解NPM基因突变在白血病发病中的分子机制。

中文关键词： 核仁磷酸蛋白；基因突变；白血病；检测；分子机制

英文摘要： NPM1 mutations have been recently identified as the most frequent genetic alteration in acute myelogenous leukemia (AML). NPM1 mutations occur in about one third of adult AML and generate NPM mutants that localize aberrantly in the leukemic-cell cytoplasm. NPM-cytoplasmic positive (NPMc+ AML) is recognized as a new provisional entity in the World Health Organization (WHO) 2008 classification of myeloid neoplasms. NPM1 mutations may become a new tool for estimating prognosis and monitoring minimal residual disease in AML. Therefore, it's necessary to establish a detection method of NPM mutations in clinical practice and further clarify the role of NPM mutants in leukemogenesis. In present study, real-time quantitative polymerase chain reaction (RQ-PCR) would be used to examine the NPM gene mutations, and the mAbs against NPM mutation A (NPM-mA) would be used to detect the cyto-plasmic dislocation of NPM in leukemia cells. To investigate the role of NPM mutations in leukemogenesis, the recombinant retroviral vector containing NPM mutation A (NPM-mA) or interfering RNA to mutant NPM gene would be transfected into leukemic cell lines respectively, soft agar colony formation assay, FCM, RT-PCR and transwell assay would be used to detect the cell proliferation, cell cycle, apoptosis, and chemotaxis capabilicy. This project could promote the applications of NPM mutations in the clinical hematology tests and help to understand the molecular mechanisms of NPM gene mutations in leukemogenesis.

英文关键词： nucleophosmin; gene mutation; leukemia; detection; molecular mechanism