BDNF/TrkB途径介导调控骨髓瘤MDSCs破骨分化的作用和机制

Lytic bone disease in multiple myeloma (MM) can impede the patient's quality of life and correlated to prognosis. Recent studies have reported that Myeloid- derived suppressor cells (MDSCs) is a novel osteoclast (OCs) progenitors in patients with osteolytic malignancies, the quantity of MDSCs were significantly increased in the bone marrow, spleen and peripheral blood of patient with active MM compared with healthy donor. Furthermore, MDSCs from myeloma-bearing mice can differentiate into mature and functional OCs in vitro and in vivo. However, the molecular mechanism regulating MM-MDSCs differentiation into OCs is still unclear. Our previous studies have found that brain-derived neurotrophic factor (BDNF) and its receptor TrkB are over-expressed in MM, and BDNF induces OCs differentiation in TrkB+ peripheral blood mononuclear cell cultures, blocking of BDNF can inhibit bone destruction and tumor growth dramatically, then we propose that BDNF may regulate the differentiation of MDSCs to OCs, and involve in pathogenesis of bone diseases. This project intends to, based on MM patients’ peripheral blood and bone marrow specimens, analyze the relationship among BDNF levels, the number of MDSCs and OCs in bone marrow, and the clinical stage; and take immune magnetic beads and flow separation method to screen the primary MM cells and MDSCs, and observe the regulation of BDNF on MDSCs homing, proliferation and osteoclast differentiation by using transwell co-culture system; establish MM in vivo models by SCID-rab and tail veil injection, analyze the regulation of BDNF on the homing and OCs differentiation of MDSCs by using shRNA interference, luciferase reportor gene, and Optical in vivo Imaging technology, and to identification of new targets for myeloma therapy.

多发性骨髓瘤（MM）骨病严重影响患者生活质量和预后。最新发现髓源性抑制细胞（MDSCs）是溶骨性肿瘤一种新的破骨细胞（OCs）前体，在MM患者骨髓、脾脏和外周血中明显扩增，MM小鼠的MDSCs体内外均能分化OCs，但MM-MDSCs分化的调控机制不明。我们前期发现MM高表达脑源性神经营养因子（BDNF）及受体TrkB，BDNF能诱导外周血单核细胞破骨分化，且阻断BDNF抑制骨质破坏，提示BDNF可能调控MDSCs破骨分化参与骨病；本项目拟选用MM患者外周血和骨髓标本，分析BDNF与骨髓MDSCs、OCs数量和临床分期的关系；免疫磁珠和流式分选筛选MMs和MDSCs，Transwell共培养研究BDNF对MDSCs破骨分化的作用和分子机制；建立SCID-rab和尾静脉注射体内模型，利用shRNA、荧光素酶标记、活体显像技术观察BDNF调控MDSCs归巢、破骨分化的作用，寻找MM防治新靶标。

多发性骨髓瘤是骨髓内恶性浆细胞异常增生的血液系统恶性肿瘤。骨髓瘤骨病严重影响患者生活质量和预后。研究发现髓源性抑制细胞（MDSCs）是溶骨性肿瘤一种新的破骨细胞（OCs）前体，在MM患者骨髓、脾脏和外周血中明显扩增，MM小鼠的MDSCs体内外均能分化OCs，但MM-MDSCs分化的调控机制不明。前期研究发现骨髓瘤MSCs参与调节MDSCs的活化和扩增；本项目拟选用MM患者外周血和骨髓标本，分析MDSCs表达模式、数量和临床分期的关系；免疫磁珠和流式分选筛选MM-MSCs和MDSCs，Transwell共培养研究MSCs对MDSCs破骨分化的作用和分子机制；建立SCID-rab和尾静脉注射体内模型，利用shRNA、荧光素酶标记、活体显像技术观察MSCs调控MDSCs破骨分化的作用和机制，寻找MM骨病治疗的新靶标。结果：1.多发性骨髓瘤患者外周血及骨髓中MDSCs均明显增高；2. MM患者MDSCs水平与ISS分期，疾病进展显著相关，与免疫球蛋白亚型无明显关联；3. 与外周血相比，MM骨髓中M-MDSCs的比例更高, M-MDSCs水平与sRANKL、sRANKL/OPG、IL-6表达水平和骨病分级呈正相关；4. MM细胞体外能促进MSCs表达和分泌VEGF、GM-CSF、sRANKL、sRNAKL/OPG、TNFα、IL-6、IL-10等细胞因子；5. 体外MM-MSCs能通过表达VEGF、GM-CSF、M-CSF、S100A8/A9、TNF、IL-6、IL-10诱导MM-MDSCs扩增和活化；6. MM-MSCs通过上调RNAKL促进M-MDSCs的NF-kB和NFATc1信号途径激活，从而促进M-MDSCs向破骨细胞分化。7. MM-MSC通过RANKL/NFATc1途径上调M-MDSCs中 TRAP、cathepsin K、MMP-9的表达促进骨吸收。8. RANKL抑制剂（Denosumab）能阻断MSC和M-MDSCs间的相互作用，抑制M-MDSCs的扩增和活化，抑制TRAP酶的表达和溶骨活性。9. Denosumab能显著降低MM小鼠模型体内M-MDSCs的水平，减轻小鼠模型的骨质破坏程度和骨矿物质密度（BMD），抑制小鼠模型肿瘤生长并延长生存。