SDF-1诱导椎间盘干细胞niche内多能干细胞迁移的效应机制及其纳米微粒原位诱导组织再生作用的研究

Situ induced tissue regeneration can promote intervertebral disc (IVD) self-repair, and does not depend on exogenous stem cells, but it is difficult in recruitment endogenous stem cells in situ in the IVD, which is absence of blood vessels. However, self-regeneration mechanism of the IVD to further clarify will help solve this problem. Our previous studies have shown that SDF-1, an important stem cell migration factor, express significantly higher in degenerative IVD than normal group, and SDF-1 implanting can relieve the degeneration of nucleus pulposus in rat tail degeneration model. Resent researches found a potential stem cell niche in a region close to the IVD in both humans and a variety of animals including mice, rabbits, and pigs. This niche is located in the regions adjacent to the disc, such as the perichondrium and the annulus fibrosus ligament anchoring site. Rat stem cells in this niche (ISN-SCs) were successfully isolated and it was confirmed that the ISN-SCs met the minimal criteria for the definition of multipotent mesenchymal stromal cells, and had a multipotent differentiation potential. In particular, ISN-SCs showed a greater potential for osteogenesis and chondrogenesis. Studies also indicated that cellular migration had taken place from the stem cell niche areas into the mature cartilaginous tissues of the IVD. However, the ubiquity and mesenchymal stem cell characteristics of mammalian ISN-SCs are needed to be identified. Furthermore, the effect mechanism of SDF-1 inducing stem cells migration in IVD stem cell niche, and the biological effects of ISN-SCs in the IVD are both unclear. On the basis of our previous works, our study is divided as followed: (1) A variety of mammalian ISN-SCs are isolated and identified; (2) SDF-1 bovine serum albumin nanoparticles are constructed; (3) Study on effect mechanism of SDF-1 inducing ISN-SCs migration from the stem cell niche into the IVD in vitro, using transwell culture system and disc organ culture model; and (4) Effect of SDF-1 nanoparticles for in situ disc tissue regeneration in animal IVD degeneration model. The results of this study will help to clear the effect mechanism of SDF-1 inducing ISN-SCs migration, and the biological effects of ISN-SCs in the IVD, reveal the self-regenerating mechanism of the IVD, and lay the foundation for new drugs for in situ IVD tissue regeneration.

原位诱导组织再生可用于促进椎间盘自我修复，但难在无血管的椎间盘中原位募集干细胞，椎间盘自我修复机制的进一步明确有助于解决难题。我们的前期研究显示，干细胞迁移因子SDF-1表达量随椎间盘退变而显著增高，内植后能延缓动物体内的髓核退变。最新研究发现，纤维环外层附着处及毗邻软骨膜内存在干细胞niche，富集多能干细胞（ISN-SCs），且不受无血管的限制而向髓核内迁移。但是，ISN-SCs受SDF-1诱导而迁移的效应机制及其在椎间盘内的生物学作用仍不明确。本项目拟在前期工作基础上，鉴定哺乳动物ISN-SCs的普遍存在性及干细胞特性，构建SDF-1白蛋白纳米微粒，运用细胞迁移、离体椎间盘器官和椎间盘退变模型等技术，阐明SDF-1诱导ISN-SCs迁移的效应机制，明确ISN-SCs在椎间盘内的生物学作用，和SDF-1纳米微粒的原位诱导组织再生作用，揭示椎间盘自我修复机制，为研发新型复合药物奠定基础。

原位诱导组织再生可用于促进椎间盘自我修复，但难在无血管的椎间盘中原位募集干细胞，椎间盘自我修复机制的进一步明确有助于解决难题。我们的前期研究显示，干细胞迁移因子（stromal cell de-rived factor-1,SDF-1）表达量随椎间盘退变而显著增高，内植后能延缓动物体内的髓核退变。最新研究发现，纤维环外层附着处及毗邻软骨膜内存在干细胞niche，富集多能干细胞（ISN-SCs），且不受无血管的限制而向髓核内迁移。但是，ISN-SCs受SDF-1诱导而迁移的效应机制及其在椎间盘内的生物学作用仍不明确。本项目通过研究成功鉴定出多种哺乳动物ISN-SCs的普遍存在性及干细胞特性，成功构建SDF-1白蛋白纳米微粒，运用细胞迁移实验阐明SDF-1诱导干细胞迁移进入椎间盘的效应机制。通过椎间盘退变模型，在大鼠尾部的椎间盘制造缺损模型，注射SDF-1和负载SDF-1的纳米微粒，证实能显著促进椎间盘中纤维环的修复，验证SDF-1纳米微粒的原位诱导组织再生作用。在研究中，我们进一步思考，椎间盘组织高渗、酸性、缺氧的环境不利于干细胞的存活，将限制干细胞治疗椎间盘退变的疗效，导致内源性修复的失败。国内外学者研究发现，在低氧微环境中，MSCs可向髓核表型分化，而缺氧时调节细胞能量代谢和存活能力的关键分子是HIF-1α。在此研究基础上，我们项目组围绕椎间盘内的HIF-1进行了相应的一系列研究。研究证实，HIF-1/SDF-1通路是调控干细胞迁移的重要通路；低氧是促进干细胞定向髓核样分化的关键条件，而HIF-1α是促进干细胞定向髓核样分化的关键基因，HIF-1α可能通过调控Notch/Twist1通路和Sonic Hedgehog通路影响干细胞定向髓核样分化。以上研究成果将进一步揭示并完善椎间盘通过内源性干细胞的自我修复机制，为研发新型复合药物实现原位诱导椎间盘组织再生奠定基础。