Crif1/VEGF-A介导巨噬细胞转分化为内皮样细胞修复造血龛损伤的机制研究

The regeneration of bone marrow sinusoidal endothelium is a critical and necessary step in reconstruction of hematopoietic niche and hematopoietic function following radiochemotherapy and nuclear accident injury, but its mechanism is not fully understood. Macrophages (MФ), which are widely distributed in the bone marrow, have the potential of transdifferentiation into endothelial cells. Our pre-research indicated that irradiation damage could up-regulate the expression of Crif1 and VEGF-A in bone marrow MФ cells, and the M2 MФ cells might transdifferentiation into novel sinusoidal endothelial-like cells (CD206+ENG+) involved in the regeneration of sinusoids by a “fill in”way. It is known that Crif1 is a STAT3-specific transcriptional coactivator. Thus we speculate that activation of Crif1/STAT3 may up-regulate VEGF-A transcriptional expression, which in turn induces M2 MФ cells transdifferentiation into endothelial-like cells involved in the reconstruction of hematopoietic niche. To verifiy the function and mechanism of transdifferentiation from M2 MФ cells into endothelial-like cells in hematopoietic reconstruction after radiation injury, we propose to design the pre-clearing and re-infusion of MФ cells in vivo experimention, and to use gene interference technology and STAT3 inhibitors to investigate Crif1/STAT3/VEGF-A signaling. The completion of this project will further reveal the cellular and molecular mechanisms of the hematopoietic reconstruction, which will lay a theoretical foundation for the new strategies of bone marrow hematopoietic failure therapy.

骨髓血窦的修复是放化疗和核事故损伤后造血龛及造血功能重建的必要且关键步骤，相关机制尚不完全清楚。广泛分布于骨髓中的巨噬细胞（MФ）具有转分化为血管内皮细胞的潜力。预研结果提示，辐射损伤诱导小鼠骨髓MФ上调Crif1和VEGF-A，且M2型MФ可转分化为新型内皮样细胞（CD206+ENG+）并“填充式”修复受损血窦。已知Crif1是STAT3特异性转录共激活因子，推测Crif1/STAT3上调VEGF-A表达，进而诱导M2型MФ转分化为内皮样细胞参与造血龛重建。本研究拟通过预清除和再输注MФ的体内实验，进一步验证辐射损伤后M2型MФ转分化为内皮样细胞参与血窦修复和造血龛重建，并利用基因干扰技术和STAT3抑制剂探讨Crif1/STAT3/VEGF-A介导M2型MФ转分化为内皮样细胞的作用机理。本项目旨在进一步揭示造血龛重建的细胞与分子机制，为探索治疗骨髓造血抑制的新策略奠定理论基础。

血窦再生是放射损伤后造血微环境以及造血重建的重要前提条件，但相关细胞分子机制尚未被完全认识。本研究以5 Gy Co-60伽玛射线全身照射建立中度骨髓型急性放射综合症小鼠模型，考察了放射损伤后骨髓巨噬细胞（BM-Mφ）的激活表型，及其在血窦再生和造血重建中的作用并探索其潜在的分子机制，得到了如下结果：1. BM-Mφ具有一定放射耐受性，存活的BM-Mφ激活，尤其是CD206+ BM-Mφ比例极大提高；清除BM-Mφ阻碍了骨髓造血干祖细胞（HSPCs）的恢复；2. 放射损伤导致骨髓血窦扩张和破裂，血窦内皮细胞（SECs）数量降低；放射损伤后BM-Mφ，尤其是激活的CD206+ BM-Mφ，上调血管内皮生长因子（VEGF-A）的表达；体内细胞示踪实验显示BM-Mφ未能直接转分化参与血窦再生，但是清除BM-Mφ显著降低骨髓总VEGF-A的蛋白水平并阻碍SECs数量和血窦结构的恢复；3. 放射损伤后骨髓虚空，存活的BM-Mφ被拉伸；BM-Mφ上调机械敏感性离子通道Piezo1的表达并响应机械拉伸刺激，激活钙离子/CaN/NFAT/HIF-1α信号通路，从而上调VEGF-A的表达。本研究揭示了BM-Mφ促进放射损伤后血管龛再生的重要功能，为探索骨髓型ARS救治新策略提供理论基础。