脂肪组织巨噬细胞（ATMs）替代激活及调控在股骨头坏死发病机制及治疗中的作用研究

Steroid-induced osteonecrosis involves a series of pathophysiological reactions, such as enhanced adipogenesis ,decreased lipid transfer, and accumulated adipocytes, resulting in avascular necrosis of bone tissue. Adipose tissue macrophages (ATMs) are the predominant leukocyte population in fat; during the process of osteonecrosis, ATMs differentiate into different functional types of cells:'Classically activated' M1 macrophages and 'alternatively activated'M2macrophages. M1 ATMs promote the process of inflammation; in contrast, M2 ATMs suppress the inflammatory response and promote enhanced damage repair. Interleukin-4 (IL-4) induces alternative activation of recruited macrophages, and granulocyte macrophage colony stimulating factor (GM-CSF),a common immune adjuvant, has a synergistic effect with IL-4. In the study, the combination of IL-4 and GM-CSF will be used to induce alternative activation of ATMs, then observe the effects on the phenotypic and functional characteristics, especially the effects on adipocyte proliferation, lipid metabolism, inflammatory factor secretion, and the effects on the osteogenic and adipogenic differentiation of mesenchymal stem cells. Through quantitative analysis of the infiltrating distribution and composition of M1 / M2 ATMs in the necrotic area, the relevance between osteonecrosis and ATMs polarization will be discussed, and the effects of alternative activation and regulation of ATMs using IL-4/GM-CSF on the prevention and treatment of osteonecrosis will be investigated. On that basis, the pathogenesis of steroid-induced osteonecrosis will be further studied, and a new treatment target for osteonecrosis will be carried out.

激素性骨坏死发病过程中，脂肪分化增强、脂质转运减弱，脂肪细胞异常堆积，激发一系列病理生理反应，导致骨组织缺血坏死。脂肪组织巨噬细胞(ATMs)在骨坏死不同阶段分化成不同功能类型细胞：经典激活巨噬细胞(M1 型)和替代激活巨噬细胞(M2 型)。M1型细胞促进炎症反应进行；M2型细胞抑制炎症反应，促进损伤修复。白介素-4（IL-4）可诱导巨噬细胞替代激活，GM-CSF与IL-4有协同作用。本研究应用IL-4/GM-CSF诱导ATMs替代激活，观察其表型及功能变化特征，尤其是对脂肪细胞增殖、脂质代谢及炎症因子分泌影响，对间充质干细胞成骨成脂分化的影响；通过对骨坏死区M1/M2型ATMs浸润分布及构成比定量分析，探讨ATMs极化差异与骨坏死发生相关性，以及IL-4/GM-CSF替代激活及调控对激素性骨坏死的防治作用；并在此基础上对激素性股骨头坏死发病机理进行深入探讨，寻找骨坏死治疗新的调控靶点。

通过建立激素性骨坏死模型，采用特异性蛋白分子F4/80, CD11c 和CD206用来鉴定M1和M2巨噬细胞；在此基础上采用流式技术、免疫组化、免疫荧光、SLISA、RT-PCR等方法，比较分析激素性骨坏死鼠外周循环M1/M2型巨噬细胞表型及功能变化，以及骨坏死区M1/M2型ATMs浸润分布与构成比的定量分析，探讨ATMs极化差异与骨坏死发生的相关性；结果显示巨噬细胞参与调控的局部免疫及炎症反应在激素性股骨头坏死的发病过程中发挥重要的作用；随着脂肪细胞不断堆积，炎症反应不断加强，病变区ATMs的浸润越来越多；坏死早期：巨噬细胞激活，以M1为主, 促炎因子后期： 巨噬细胞激活减弱， 主要呈现为M2 巨噬细胞，抗炎因子分泌IL-4干预，能明显减低M1、M2型巨噬细胞的浸润，减轻炎症反应。进一步实验联合应用IL-4/GM-CSF，替代激活ATMs，观察其生物学效应。研究发现，IL-4能够降低激素性骨坏死的发生率，减少骨坏死区破骨细胞的数目。IL-4干预可抑制TNF-α的表达，减少骨坏死区域M1巨噬细胞的浸润，且维持较高水平的M2巨噬细胞；TUNNEL检测发现IL-4可减少骨坏死区凋亡细胞的数目，其发生机制与STAT1磷酸化及caspase-3活化信号通路相关。因此，IL-4能减轻鼠激素性骨坏死的发生，其调控机制是通过STAT1-caspase-3信号通路抑制炎症反应、减低M1巨噬细胞浸润、抑制TNF-a介导的骨细胞的凋亡来实现的。