KLF4介导骨髓纤维症血液干祖细胞异常增殖的作用机制

Myelofibrosis (MF) is a rare blood disease, and often accompanied by splenomegaly, but the pathogenesis is still unclear. Our previous study found that in patients with MF, the expression of KLF4 in blood stem／progenitor cells (CD34+ cells) was significantly down-regulated, while the downstream p21 gene expression was also reduced accordingly. Given that KLF4 is a key stem cell-dependent transcription factor, we hypothesize that in the process of MF, the KLF4 promoter region in CD34 + cells is hypermethylated, resulting in the inhibition of KLFF4 expression and the down-regulation of p21 expression, so leading to CD34+ Cells multiply in a special microenvironment. Therefore, we intend to first analyze the methylation status of the KLF4 promoter region, and then isolate normal cord blood CD34 + cells. Forced expression or gene editing techniques were used to generate the CD34+ Cell line with upregulation or knock-out KLF4 gene, respectively. Then, the proliferation and differentiation status of CD34+ cells with gene expression profile will be characterized. Furthermore, through the co-culture with bone marrow mesenchymal stram cells and endothelial cells, as well as xenotransplantation into humanized bone model, the KLF4-drived abnormal proliferation of CD34+ cells was explored at the molecular, cellular and in vivo level. The project results obtained might be evidence to reveal the pathogenesis of MF and provide potential therapeutic targets to interfering the disease.

骨髓纤维化（MF）属于罕见血液疾病，至今发病机理仍不清楚。我们前期研究发现，MF患者体内血液干祖细胞(CD34+细胞)的KLF4基因表达明显下调，与此同时下游的p21基因表达也相应降低。鉴于KLF4是一种关键的干细胞干性因子，我们猜测：在MF发病过程中，CD34+细胞KLF4基因启动子区域处于超甲基化状态，导致KLF4表达抑制，诱导p21表达下调，从而使CD34+细胞在一种特殊的微环境中大量增殖。因此，我们拟首先分析KLF4基因启动区域的甲基化状态，进而分离正常脐带血CD34+细胞，利用过表达或基因编辑技术上调或敲除KLF4基因，观察分析CD34+细胞增殖／分化状态以及基因表达谱特征；进一步通过与骨髓间充质干细胞和内皮细胞共培养，以及异种移植入人源化小骨模型中进行分析，从而在分子、细胞和在体水平探究KLF4介导CD34+细胞异常增殖的作用，为揭示MF发病机制和寻找潜在治疗靶点提供依据。

骨髓纤维化（MF）是一种BCR-ABL阴性的骨髓增殖性肿瘤，属罕见慢性白血病，其根本致病原因是血液干祖细胞(CD34+细胞)的过度增殖。近十年间虽然已鉴定出了几个体细胞驱动突变，但目前MF发生发展的分子机制尚不完全清楚，其治疗手段也大多为姑息性治疗。因此，更深入地了解MF发病的复杂机制是寻找干预和阻断该病策略的必经之路。本研究发现，KLF4（Krüppel-like factor 4）在MF患者的CD34+细胞和MF模型细胞（hel92.1.7细胞、set2细胞）中被甲基化抑制，其表达显著下调。过表达KLF4可以显著抑制MF模型细胞的增殖及克隆形成能力。在机制上，KLF4的表达可以激活其靶基因RELN的转录表达，进而通过PI3K/AKT信号通路调控细胞的增殖能力。因此，本研究项目从MF患者CD34+细胞中KLF4异常表达的表型出发，探索了相应机制，构建了细胞模型。后续将基于阐明的机制探索临床干预MF诊疗的综合方案，为髓系血液疾病的诊疗提供实验证据。

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