骨髓微环境调控骨髓瘤细胞RANKL表达的作用机制研究

项目名称： 骨髓微环境调控骨髓瘤细胞RANKL表达的作用机制研究

项目编号： No.81470369

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 詹楚生

作者单位： 香港大学深圳研究院

项目金额： 75万元

中文摘要： RANKL是导致骨髓瘤骨病发生的重要因子,研究显示其表达受启动子区甲基化的调控。我们前期研发现骨髓瘤细胞WL-2和LP-1的RANKL启动子呈完全甲基化，5-AzadC可导致它们发生去甲基化和RANKL表达上调，MSC与骨髓瘤细胞株transwell共培养可使细胞RANKL发生去甲基化并上调RANKL表达。本项目拟在前期研究的基础上，采用MSC与WL-2或LP-1共培养，检测多种去甲基化蛋白的变化，并采用多种MSC分泌的细胞因子干预两种骨髓瘤细胞，探讨何种细胞因子导致骨髓瘤细胞RANKL发生去甲基化。同时采用骨髓瘤SCID/Hu小鼠模型，观察体内微环境对骨髓瘤细胞RANKL甲基化和表达的影响，确证微环境通过去甲基化机制调控骨髓瘤细胞RANKL表达。本项目分别通过体外和体内模型，探讨骨髓微环境调控骨髓瘤细胞RANKL表达的分子机制，为探索骨髓瘤骨病的发生开辟新思路和新途径。

中文关键词： C17_骨髓瘤；间充质干细胞；核因子κ；B受体活化因子配体；甲基化

英文摘要： RANKL play a pivotal role in the development of osteolytic bone lesions in multiple myeloma. On the other hand recent data shows that RANKL expression is regulated by promoter methylation. We aimed to study the regulation of RANKL expression in myeloma cells by promoter methylation. We first determined the methylation status of RANKL promoter in 10 human myeloma cell lines using methylation-specific polymerase chain reaction (MSP). We found that RANKL promoter was completely methylated in WL-2 and LP-1. Hypomethylation treatment (5-AzadC) of WL-2 and LP-1 myeloma cells led to demethylation of RANKL promoter and upregulation of RANKL expression. Furthermore, co-culture of the normal bone marrow-derived mesenchymal stromal cells (MSC) with WL-2 or LP-1 myeloma cells in a transwell system for 6 days resulted in repression of DNA methyltransferase-1 (DNMT-1), demethylation of RANKL and upregulation of RANKL expression in WL-2 or LP-1 myeloma cells. To clarify the mechanism involved in the demethylation of myeloma cells, demethyaltion related protein expression in myeloma cells will be determined after transwell coculture of MSC and WL-2 or LP-1. The cytokines secreted by MSC will be used to treat myeloma cells directly to explore which cytokines is involved the demethylation mechanism. Meanwhile, to discover the bone marrow microinviroment effect on myeloma cells RANKL myethyaltion and expression, the subcutaeous transplantated fetal bone will be injected with WL-2 or LP-1 cells in the SCID/Hu mouse myeloma modle, and the CD138+ myeloma cell will be separated through microbead method and the RANKL methyaltion and expression will be determind. The project devotes to reveal the new mechanism of myeloma bone lesion and find the new target for treatment of myeloma bone lesion.

英文关键词： myeloma;Mesenchymal stem cells;RANKL;methylation