S1P-S1PR2轴介导Muse干细胞归巢促进脊髓损伤修复的机制研究

干细胞移植为脊髓损伤治疗带来新希望。既往研究表明静脉移植的Muse干细胞可自主归巢于损伤组织、定向分化为受损细胞重建功能，其归巢机制可能是S1P-S1PR2轴介导。我们已证明Muse干细胞可诱导分化为NSC，局部注射后其可向脊髓损伤中心区迁移，并促进大鼠运动功能恢复。而静脉移植的Muse干细胞是否受S1P-S1PR2轴调控归巢并促进神经修复尚不清楚。本项目拟于体外应用转录组测序技术揭示Muse干细胞神经营养因子和S1PR2受体等基因表达水平，应用受体拮抗剂/激动剂明确S1P-S1PR2轴调控Muse干细胞迁移的机制。体内研究通过液相色谱和免疫组织荧光染色等分生技术、DTI影像和BBB评分等方法揭示Muse干细胞通过S1P信号归巢于脊髓损伤区并向神经细胞分化，且通过调控TNF-α/IL-10炎症因子水平改善微环境促进脊髓损伤修复。最终为Muse干细胞移植治疗脊髓损伤的临床应用提供理论基础。

Spinal cord injury (SCI) is normally disastrous for a family. Despite numerous therapy strategies, specific effective medicine or methods for SCI are not available worldwide yet. However, stem cells transplantation is a very promising alternative to treat the patients with SCI. Multilineage-differentiating Stress-Enduring (Muse) cells were discovered in 2010 as a subpopulation of mesenchymal stromal cells (MSCs). Muse stem cells can self-renew and tolerate severe culturing conditions. These cells can differentiate into three lineage cells spontaneously or in induced medium but do not form teratoma in vitro or in vivo. As a novel kind of pluripotent stem cells, Muse cells have shown great regeneration capacity in many animal models, including acute myocardial infarction, hepatectomy, acute cerebral ischemia and so on. Furthermore, the differentiation of Muse stem cell did not need to be induced before the transplantation and no tumors were observed in eight months after the transplantation. After administrated by intravenous injection, Muse stem cells can home to the injury site, survive, migrated, and differentiate into target injured cells and contribute to the recovery of function. Prof. Mari Dezawa, the scientist who discovered the Muse stem cells, had demonstrated that the Muse stem cells homed to the acute myocardial infarction by S1P-S1PR2 axis in rabbit. However, whether Muse stem cells administrated by intravenous could home to the injured spinal cord by S1P-S1PR2 and improve the neurorestoratology and functions deserves to be explored..In vitro, we have successfully used magnetically activated cell sorting (MACS) to isolate SSEA3+ cells to obtain Muse stem cells. The expressions of SSEA3, CD105, CD90, CD73, CD45 and CD14 are measured by Flow Cytometry to identify Muse stem cells. Then in this project we will use the single-cell RNA-sequencing technique to reveal Muse cells’ gene profile, especially the gene expressions of neurotrophic factors, such as NGF, BDNF, NT3, GDNF, and LIF. The scRNA-seq will also reveal the gene expression of S1PR2 of the Muse stem cells. The following procedure is to demonstrate the S1P-S1PR2 axis mediates the migration of Muse stem cells in a Boyden Chamber with the JTE-013 (antagonist of S1PR2 receptor) or CYM-5520 (agonist of S1PR2 receptor)..In vivo, we will use the Sprague-dawley rats to make the standard spinal cord injury models, and administrate the GFP+Muse stem cells/PBS into the caudal vein of the rats one day after the SCI, with 1 million of cells per kilogram of the rat. The observation period is 8 weeks. Liquid chromatography technique will be conducted to test the concentration of S1P of the rat plasm. We use the BBB scores and Keck Center autophagia scores to evaluate the function recovery. Other methods include MEP/SEP neuroelectrophysiological examination, Human hMit, and inflammatory factors (TNF-α/IL-10). For the fixed samples, human MAP2, NeuN, GFAP，5-HT and GST-pi will be analyzed by immunohistochemistry. The final goal is to evaluate the safety and efficiency of Muse stem cells transplantation in the treatment of spinal cord injury.