目的：骨髓增殖性肿瘤（myeloproliferative neoplasms，MPNs）是因造血干细胞（hematopoietic stem cells，HSCs）水平的突变而引起的一类克隆性疾病，其主要包括费城染色体阴性MPN（Ph^-MPN）的真性红细胞增多症（Polycythemia vera，PV）、原发性血小板增多症（essential thrombocythemia，ET）和原发性骨髓纤维化（primary myelofibrosis，PMF）以及费城染色体阳性的慢性粒细胞白血病（chronic myelocytic leukemia，CML）等疾病。对于Ph^-MPN，既往研究大多聚焦于疾病后期，即发生MF后的病理改变，而对于疾病进展过程中的微环境细胞的变化，尤其是成骨的变化规律，目前并不多见。本课题利用JAK2^V617F转基因小鼠模型和患者样本，对MPN不同进展阶段造血微环境中各种细胞尤其是成骨的变化规律进行了初步探索，以期为探讨MPN的发病机制提供新的视角，也为进一步治疗MPN提供实验和理论依据。

方法：首先，我们利用JAK2^V617F转基因小鼠模型进行了下列实验：每月定期检测对照组野生型（wild type, WT）小鼠和JAK2^V617F小鼠血常规；选取2、8和12 M的小鼠处死后取肝脾称重，取脾和股骨进行HE和网状纤维染色，外周血涂片并统计小鼠发病情况；流式细胞术检测2、8和12 M的 WT和JAK2^V617F小鼠MSC、EC、OBC细胞比例；根据骨髓HE染色计算不同月龄WT和JAK2^V617F小鼠骨小梁面积比例；half bone免疫荧光染色检测不同月龄WT和JAK2^V617F小鼠OBC、EC细胞比例；流式细胞术检测WT和JAK2^V617F小鼠MSC表面标志物；成纤维细胞集落形成实验（colony forming units-fibroblast, CFU-F）检测2 M和8 M的WT和JAK2^V617F小鼠MSC CFU-F形成能力；SRB实验检测2 M和8 M的WT和JAK2^V617F小鼠MSC体外增殖能力。其次，我们建立共移植MPN嵌合小鼠模型，流式细胞术分析移植小鼠OBC比例。再次，我们分选2 M和8 M的WT和JAK2^V617F小鼠骨髓间充质干细胞（mesenchymal stem cells, MSCs）进行转录组测序，并对转录组结果进行GO、KEGG和GSEA分析。最后，我们通过流式细胞术检测健康供者（healthy donor, HD）和JAK2^V617F患者MSC表面标志物；通过数据挖掘分析HD和JAK2^V617F患者MSC转录组差异；EdU实验检测HD和JAK2^V617F患者MSC的细胞周期；SRB实验检测HD和JAK2^V617F患者MSC增殖能力；β-半乳糖苷酶染色实验检测HD和JAK2^V617F患者MSC衰老比例；细胞分化实验检测HD和JAK2^V617F患者MSC成骨改变；HE和网状纤维染色检测HD和JAK2^V617F患者骨髓成骨和成纤维变化；鹅卵石区形成细胞实验（CAFC）及长周期起始细胞培养实验（LTC-IC）用来评估HD和JAK2^V617F患者MSC的造血支持能力及共培养后造血干/祖细胞的功能。

结果：首先，JAK2^V617F转基因小鼠模型结果提示，2 M的JAK2^V617F小鼠HGB、HCT、PLT数值明显增高，显示出类似MPN发病的症状；在疾病的早期（2 M），JAK2^V617F小鼠肝脾大小与正常WT小鼠相比无统计差异，但是随着疾病进展，逐渐展示出肝脾肿大、股骨发白等特征。JAK2^V617F小鼠脾和股骨均有巨核细胞增多、纤维化加重等病理变化；且直到8 M才有部分JAK2^V617F小鼠发生了骨髓纤维化，发病进程缓慢，符合临床中常见的MPN发病规律。流式结果提示，8 M时，JAK2^V617F小鼠骨髓中MSC、EC比例升高。流式细胞术、HE染色、half bone免疫荧光染色结果提示，2 M的 JAK2^V617F小鼠OBC比例低于WT小鼠，但在8 M的JAK2^V617F小鼠OBC比例高于WT小鼠。双光子成像结果显示，在2 M的JAK2^V617F小鼠骨胶原含量明显低于WT小鼠；但在8 M的JAK2^V617F小鼠骨胶原含量明显高于WT小鼠。CFU-F实验证实，2 M的JAK2^V617F小鼠MSC CFU-F形成能力较强；然而与同月龄的WT小鼠CFU-F结果相比，8 M的JAK2^V617F小鼠MSC CFU-F形成能力较弱。SRB实验结果提示，与同月龄WT小鼠相比，2 M、8 M的JAK2^V617F小鼠MSC增殖无差异。其次，共移植MPN嵌合小鼠模型显示，移植后两个月 JAK2^V617F/GFP共移植组小鼠OBC细胞比例明显低于WT共移植组。再次，转录组测序结果提示，2 M的JAK2^V617F小鼠 MSC促髓系细胞发育相关通路显著富集；骨重塑能力增强，成骨分化通路富集减弱；炎症相关的通路显著富集；增殖、血管形成相关通路显著富集。8 M的JAK2^V617F小鼠 MSC促髓系细胞发育相关通路富集减少；骨硬化能力增强；血管形成相关通路上调；炎症相关通路有富集减少的趋势，而抗炎因子IL-4通路富集增加。最后，临床样本相关实验证实，JAK2^V617F患者的MSC增殖加快，衰老比例显著低于HD MSC，成骨能力增强，成骨分化通路显著富集，而炎症相关通路IFN-γ、TNFα、IFN-α富集减少，促血管生成相关通路上调。随着骨髓纤维化程度的增加，JAK2^V617F患者骨髓成骨比例增加。CAFC和LTC-IC实验提示，JAK2^V617F患者MSC的造血支持能力降低。

结论：本研究结果提示，MPN发病早期骨髓成骨减少，发病后期成骨增加。MPN发病不同阶段微环境细胞，尤其是MSC已经被改变。Ph^-MPN的演化对微环境具有重塑作用。

Objective Myeloproliferative neoplasms (MPNs) are a group of chronic neoplasms characterized by constitutive hematopoiesis activation of hematopoietic stem cells (HSCs), including polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF) and chronic myelocytic leukemia (CML). While PV, ET and PMF belong to Philadelphia negative myeloproliferative neoplasms (Ph^-MPNs), with JAK2^V617F as a common driver mutation. Previous studies have highlighted that malignant HSCs compete with their normal counterparts for niche resources and occupancy, while normal BM niche being disrupted. However, functional changes of “remodeled” niche are still lacking experiment evidence in MPN. Moreover, as former studies focused on osteogenesis of MF, the effect of PV and ET on bone, no matter in human or mouse, is still unclear. In our study, JAK2^V617F mutant mice and clinical samples were used to elucidate the “remodeled” niche and osteogenesis change in the progression of MPN, which may shed light on underlying mechanisms and theoretical basis of MPN disease.

Methods JAK2^V617F mice were conducted the following experiment: the peripheral blood (PB) counts of WT and JAK2^V617F mice were measured monthly. At 2 month (M), 8 M and 12 M points, the liver and spleen were weighed, meanwhile the spleen and bone were sectioned and stained with HE and reticular fiber staining. PB smear was also performed. The proportion of MSC, EC and OBC were analyzed by FACS at different time points. Trabecular bone area，OBC and EC staining and collagen areas were evaluated with HE staining, half bone immunofluorescence and two-photon microscopy in vivo, respectively. The markers of MSC in WT and JAK2^V617F mice were analyzed with FACS. CFU-F (colony forming unit-fibroblasts) assay was used to evaluate the stemness of MSC, and sulforhodamine B (SRB) staining was used to detect cell proliferation in vitro. Moreover, a co-transplanted MPN chimeric mice model was established to confirm the OBC proportion change 2 M after transplantation. Futhermore, RNA-sequencing with sorted MSC in 2 M and 8 M time points, and subsequential GO, KEGG and GSEA analyses were conducted to explore the underlying mechanisms. At last, we also used FACS to evaluate the surface markers of human MSC. Data mining was screened for RNA differences of MSC in HD and JAK2^V617F patients. EdU assay, SRB staining and β-gal staining were used to analyze the cell cycle, proliferation and senescence of human MSC, respectively. Osteogenic differentiation was conducted to evaluate the differentiation capacity of MSC in vitro, while HE and reticular fiber staining were used to confirm the osteogenesis and myelofibrosis in vivo. We also evaluated the hematopoietic support capacity of human MSC using CAFC and LTC-IC assays in vitro.

Results PB analyses showed that improved levels of HGB, HCT and PLT in JAK2^V617F mutant mice, which exhibited PV- or ET- like syndrome. Compared with WT, the liver and spleen of JAK2^V617F mice showed no statistical difference in early stage of MPN, but larger in later stage. The pathological results also indicated that, the spleen and bone exhibited increased megakaryocytes and fibrosis in later stage. Myelofibrosis began to present in bone at 8 M point, and that slow pathogenesis process was in common with clinical practice. Compared with WT, JAK2^V617F mice also exhibited higher proportion of MSC and EC in BM at 8M point. While the proportion of OBC and collagen area was less at 2 M point, but more at 8 M point in JAK2^V617F mice. Compare with MSC from WT mice of the same age, MSC from 2 M JAK2^V617F mice showed enhanced capacity of CFU-F, while the capacity was weakened in 8 M JAK2^V617F mice. But MSC from WT or JAK2^V617F mice of same age showed no difference in proliferation in vitro. In accordance with FACS results of JAK2^V617F mice, the proportion of OBC was less in co-transplanted MPN chimeric mice model 2 M after transplantation. Subsequent RNA-sequencing results implied that improved capacities of regulating myeloid cell development, angiopoiesis and bone remodeling, enhanced inflammation signaling pathway in MSC of JAK2^V617F mice at 2 M point. On contrast, at 8 M point, MSC of JAK2^V617F mice exhibited impaired capacities of regulating myeloid cell development and inflammation, while the enrichment of osteogenic differentiation and angiopoiesis augmented. Moreover, MSC from JAK2^V617F patients exhibited improved proliferation, diminished senescence, enhanced osteogenic differentiation, decreased inflammation pathway and upregulated capacity of promoting angiopoiesis. With myelofibrosis progress, the proportion of osteogenesis was increased in JAK2^V617F patients. CAFC and LTC-IC assays suggested MSC showed impaired hematopoietic support activity in JAK2^V617F patients.

Conclusion The proportion of BM osteogenesis was diminished in early stage of MPN and increased in the later stage. Microenvironment, especially MSCs, have been altered in different stages of MPN pathogenesis. The heterogeneous evolution of Ph^-MPN has a remodeling effect on the microenvironment.