白血病环境中骨髓niche成骨细胞表观遗传学研究

Bone marrow microenvironment (niche) plays a critical role in the pathogenesis of leukemia. Although abundant studies have demonstrated that niche has been closely associated with ontogenesis of leukemia, however, in luekemia stress the niche's dysfuction and its definite mechanisms are still unknown. In previous study, we have defined the dysfuction of bone marrow stromal cells from luekemic mice and acute myeloid luekemia (AML) patients. The epigenetic defects in the bone marrow stromal cells have also been demonstrated in our previous study. In present project, we would further investigate the biologic functions of osteoblasts, one of pivotal components in the bone marrow niche, with the well-established MLL-AF9 AML (MA9-AML) mouse model. The epigenetic alterations of osteoblasts in the leukemia host will be investigated through ChIP-on-Chip, and further analyzed with bioinformatics. The definitive epigenetic discrepancies will be verified with molecular and cellular biologic measures in vivo and in vitro. The impacts of definitive epigenetic alterations on the biological characteristics of leukemic stem cells (LSCs) and leukemia progression would be studied through co-cultured and co-transplanted with osteoblasts with modified with lentivirus vector along with LSCs. The available small molecular would be employed to modify the epigenetic changes in vivo. The project will specify the epigenetic alterations of osteoblats in the leukemia host, and its impacts on the biology characteristics of LSCs, and may provide evidences for the novel target of niche in leukemia therapy.

骨髓niche功能缺陷对白血病的发生发展进程具有重要影响，但白血病环境中骨髓niche细胞发生怎样的缺陷及其机制目前尚不清楚。我们前期研究发现白血病环境中骨髓基质细胞功能缺陷，而且存在甲基化表观遗传学改变。因此，本课题以建立的MLL-AF9急性髓细胞白血病（MA9-AML）小鼠为模型，观察骨髓成骨细胞生物学功能改变；采用高通量ChIP-on-Chip检测方法和生物信息学对成骨细胞进行检测和分析，筛选特异表达的表观改变并采用分子生物学和细胞生物学手段对特异表观改变鉴定；构建干预特异表观改变的慢病毒载体，采用修饰的成骨细胞与白血病干细胞（LSCs）体外共培养和体内共移植实验明确其对LSCs生物学特性及对白血病发展和小鼠生存的影响。以期通过本课题的研究，明确白血病环境中骨髓niche成骨细胞表观遗传学改变及特异表观改变对LSCs生物学特性的影响，为从niche角度治疗AML提供新思路。

骨髓造血微环境（niche）对正常造血干细胞（HSC）调控至关重要；白血病状态下可以重塑骨髓微环境，那么白血病状态下骨髓niche可能发生改变，但发生了怎样的改变？尤其是骨髓niche中主要细胞成分成骨细胞、血管内皮细胞如何改变以及改变的机制和这些改变对LSCs的影响等目前尚缺乏深入研究。我们通过非照射方法建立MLL-AF9急性髓细胞白血病（MA-AML）小数模型用于骨髓niche研究；确立1*10^3剂量白血病细胞为该研究白血病模型剂量。建立骨髓niche成骨细胞分选方法，分选标记为CD45-Lin-CD31-Sca-1-CD51+，流式细胞分选细胞纯度90%以上；在白血病细胞接种小鼠后0天、7天、14天、21天不同时间点检测骨髓成骨细胞比例，发现随着白血病进展成骨细胞比例无明显变化；但细胞数量呈减少趋势；骨髓病例检测也提示骨髓niche结构发生破坏，成骨细胞数量呈下降趋势。选取接种14小鼠模型骨髓niche成骨细胞做RNAsequence分析，结果发现与D0天相比，D14天小鼠细胞上调基因为5802个，下调基因为5428个。从中我们挑选出了41个差异度较高的基因，发现PIK3CA、Cdc42与促进肿瘤细胞浸润有关；Ppp1r12a、Prkca与白血病细胞耐药有关；Flt1与成骨细胞分化有关，且有文献指出在ALL的骨髓微环境中可发现Flt1出现表观遗传学改变；Ctbp2与ALL细胞表观遗传改变有关，且与骨髓niche的变化也存在联系；Chd8与肿瘤细胞表观遗传改变有关；Cadm1、Ccr6、Cxcl16、Tnfsf15与白血病及成骨细胞的分化均有关。通过定量RT-PCR的方法对27条差异显著基因进行验证，发现Flt1、PIK3CA低表达，Ctbp2、Cdc42、Chd8高表达。选取表达量显著变化的高表达Ctbp2和低表达Flt1基因进一步体内外研究。结果表明白血病导致骨髓niche成骨细胞发生变化，可能对白血病的发生发展具有调控作用。