SDF-1α基因转染BMSCs动员内皮祖细胞治疗兔激素性股骨头坏死

Osteonecrosis of femoral head (ONFH) is characterized with progressive course and high disability. Various treatments can be used among which stem cell transplantation is considered as the most promising biological method. Generally, local transplantation of osteogenous seed cells and stimulative revascularization are believed to be the key points in stem cell transplantation for ONFH. Bone marrow mesenchymal stem cells (BMSCs) represent a highly reliable candidate for cell-based therapeutic approaches. Our previous studies demonstrated that endothelial progenitor cells (EPCs) could be rapidly mobilized from bone marrow and recruited to accelerate the repair of damaged vascular endothelium through the SDF-1α/CXCR4 biological axis. In this study, BMSCs are transfected with SDF-1α and transplanted for treating steroid-induced ONFH in rabbits. SDF-1α transgenic BMSCs can differentiate into osteoblast and at the same time express SDF-1α continually. Then, autologous EPCs are mobilized and recruited to initiate rapid neovascularization and therefore promote osteogenesis in the necrotic lesion through the SDF-1α/CXCR4 biological axis. RT-PCR and Western blot are used to detect the changes of proliferation and differentiation of BMSCs after SDF-1α gene transfection. In vivo differentiation and survival are confirmed using immunofluorescence and immunohistochemistry. Bone regeneration and revascularization of the necrotic area of the femoral head are evaluated with the assistance of X ray, MRI, and Micro-CT scanning.

股骨头坏死致残率高，缺乏满意治疗方案，干细胞移植是一种很有前景的生物学疗法。细胞移植治疗股骨头坏死有两个关键点：提供可靠的成骨种子细胞；促进坏死区域的血管生成。BMSCs是目前移植效果较为肯定的种子细胞。我们在前期工作中证实，借助SDF-1α/CXCR4生物轴可快速动员并募集EPCs加速损伤血管内皮修复。在此基础上，本项目建立兔激素性股骨头坏死模型，将SDF-1α基因转染的BMSCs植入股骨头坏死区，修饰后的BMSCs可分化为成骨细胞，修复坏死区骨组织，并持续分泌SDF-1α，通过SDF-1α/CXCR4生物轴快速动员并自动俘获EPCs，促进坏死区再血管化，使成骨与血管生成同步进行。拟采用RT-PCR、Western blot等观察基因修饰对BMSCs增殖分化的影响，应用免疫荧光技术追踪移植细胞的分化及转归, 借助Micro-CT骨扫描、血流灌注技术评价坏死部位骨组织修复及血管再生情况。

激素性股骨头坏死治愈率低，患者生活质量低。治疗激素性股骨头坏死有两个关键点：维持坏死区域血供，保证坏死区成骨活动。前期实验证实，借助SDF-1α/CXCR4生物轴可快速动员并募集EPCs加速损伤血管内皮修复。在此基础上，以SD大鼠激素性股骨头坏死模型为研究对象，本课题主要开展以下研究内容：（1）在股骨头坏死区域植入转染SDF-1α的骨髓间充质干细胞（SDF-1α-BMSCs），检测坏死区域SDF-1α的表达，是否可以通过SDF-1α/CXCR4生物轴快速募集EPCs至坏死区域，促进坏死区血管新生，使坏死区域成骨与血管生成同步进行；（2）沉默BMSCs中miR-137-3p的表达，检测沉默后RUNX2和SDF-1α的表达及成骨潜能，观察miR-137-3p沉默后BMSCs对ONFH的修复作用；（3）沉默BMSCs中生长分化因子（GDF11）的表达，沉默后检测骨髓间充质干细胞的成骨潜力，评估其对ONFH的修复价值；（4）收集和提取富含miR-26a的人外周血CD34+干细胞来源的外泌体（miR-26a-CD34+-Exos）对股骨头坏死区域的成骨和内皮损伤的抑制作用，同时促进坏死区域的成骨作用和血管生成活性。通过上述研究可得出以下结论：（1）植入SDF-1α-BMSCs至坏死区域可以同时促进成骨和血管生成，保护股骨头，避免坏死；（2）沉默miR-137-3p可以促进Runx2和SDF-1α的表达，然后促进股骨头坏死区域的成骨作用，并动员内皮祖细胞进入外周血以修复受损的血管并促进血管的血运重建坏死区；（3）沉默生长分化因子（GDF11）的表达可增强BMSCs的成骨潜能，可有效促进大鼠股骨头坏死区的成骨，并促进股骨头坏死的修复；（4）miR-26a-CD34+-Exos可以增强成骨作用并减弱内皮细胞损伤，并促进股骨头的成骨活性和血管生成。该项目的研究成果丰富了激素性ONFH的处理方案，并提供了理论依据。