不同氧分压下Runx2信号轴与SDF-1信号轴的交叉对话对静脉移植的MSCs定向归巢效应的影响

On the base of primary study, this research investigates the time-effect relationship between oxygen pressure and femoral head necrosis and MSCs homing effect, so as to understand the mechanism and regulatory factors of MSCs homing and femoral head necrosis healing, therefore provide the basis for designing the therapeutic schedule of ANFH. On the other hand, we hope to understand the mechanism of strengthen effect on MSCs homing via cross talk between Runx2 and SDF-1 signaling axis under different oxygen pressure, to provide the basis for treatment ANFH through using MSCs.Firstly, through using Real-time PCR, Western blotting, laser scanning confocal microscopy, flow cytometry, radiological imaging and histologic methods, we will observe not only MSCs' proliferation, osteogenic differentiation and migration capacity,but also expression of Runx2 signaling axis and SDF-1 signaling axis under different oxygen concentration/pressure. Then through transfecting or antagonist activated or block the cross talk between Runx2 and SDF-1 signaling axis, we attend to identify the mechanisms of strengthen effect on MSCs homing via cross talk between Runx2 and SDF-1 signaling axis under different oxygen concentration/pressure, so as to provide more powerful experimental basis for the application of MSCs in the treatment of ANFH.

本课题拟在前期研究基础上，探讨氧分压与股骨头坏死程度、MSCs定向归巢效应之间的时效关系，以深入了解影响MSCs定向归巢效应和股骨头坏死修复的调控因素及其作用机制，为制定针对性的ANFH治疗方案奠定基础。另外，期望了解Runx2信号轴和SDF-1信号轴的交叉对话增强不同氧分压下MSCs归巢效应的作用机制，为更好地应用MSCs治疗ANFH奠定基础。实验综合运用实时荧光定量PCR、Western blotting分析、激光共聚焦显微镜、流式细胞仪、影像学及组织学等方法，在体内外观察不同氧浓度/氧分压下MSCs增殖、成骨分化、迁移能力及Runx2信号轴和SDF-1信号轴表达；再通过基因转染或拮抗剂激活或阻断两个信号轴的交叉对话，阐明两个信号轴的交叉对话增强不同氧浓度/氧分压下MSCs归巢效应的作用机制，为应用MSCs治疗ANFH提供更有力的实验依据。

由于股骨头坏死后在骨坏死局部氧分压极低，MSCs在这种极度低氧环境下增殖能力和成骨分化能力都明显下降。前期实验中发现Runx2基因能促进MSCs向骨坏死区归巢并维持较强的成骨分化能力。因此，本项目将在体内外观察激活或阻断Runx2成骨信号轴和SDF-1迁移信号轴对MSCs归巢效应的影响，实验结果显示：①不同氧分压下，单一激活Runx2成骨信号轴或SDF-1迁移信号轴都能引起这两个信号轴出现变化。而随着氧分压下降，两个信号轴的变化越明显；②不同氧分压下，同时激活或抑制Runx2成骨信号轴和SDF-1迁移信号轴能引起这两个信号轴出现更加明显变化，两种作用具有协同性。氧分压下降这种协同性仍然存在；③股骨头内氧分压越低，股骨头坏死后形态变化越大；④ANFH模型体内，激活Runx2成骨信号轴或SDF-1迁移信号轴都能促进MSCs更多地向骨坏死区归巢；⑤同时激活Runx2成骨信号轴和SDF-1迁移信号轴能促进坏死股骨头更快地修复。综上实验结果表明，Runx2成骨信号轴和SDF-1迁移信号轴在MSCs迁移和成骨分化过程中存在着交叉点，两个信号轴能协同促进MSCs归巢效应，该结果为临床中应用MSCs治疗ANFH提供了夯实的实验基础。