人嗅粘膜球状/水平基底细胞及OM-MSCs在OM-OECs治疗SCI中的协同作用机制研究

Spinal cord injury (SCI) is one of the most difficult regeneration problems in the world. Cell transplantation is proved to be a promissing alternative for this condition. Olfactory ensheathing cells (OECs) transplantation has been demenstrated, in both basic and clinical research, to be safe and effective in SCI. However, up to now the OECs are mainly obtained from the allosome olfactory bulb (OB). Effect of cells from autologous olfactory mucosa (OM) on the treatment of SCI is attacting more and more attention now..This research focuses on four kinds of cells in OM namely Globose Basal Cells (GBCs), Horizontal Basal Cells (HBCs), Mesenchymal Stem Cells (MSCs) and Olfactory Ensheathing Cells (OECs). We firstly use Neurotrophin 3 (NT-3) or Transforming Growth Factor α (TGF-α) to culture and purify the OM-OECs from the OM mixed cells and then sort them out using the Flow cytometry (Fluorescence-activated cell sorting, FACS) according to the cell surface antigen (CSA) such as P75-NRT and GFAP. Because the other three kinds own stem cell properties, they are directly sorted out from the OM mixed cells according their own CSA (CD54 for HBCs, GBC-2 and BC-Ⅲ for GBCs and CD44 and CD105 for OM-MSCs, respectively). Furthermore, we will co-culture two kinds of cells, that is, one is OM-OECs and the other is GBCs, HBCs or OM-MSCs. So we could uncover the internal mechanism between the three kinds of cells and the OM-OECs and then go out the animal experiment. The animal experiments will be conducted using the spinal cord contusion model on the SD rats. These results will be compared with the OB-OECs. So we could confirm the different effects of the four kinds of cells on SCI. So far, we preliminarily found NT-3 could benefit the purification of OM-OECs which, however, need more efforts to confirm. And when co-cultrued, OM-MSCs could highlight the viability of OM-OECs extending longer protuberances while OM-OECs could activate the OM-MSCs differentiation to neural cells. Although we make hypothesis that they could affect each other through the cell direct contact or the active factors, the mechanism needs to be explore more. We will perform the Real-time PCR to quantitate the NT-3 gene expression to evaluate the OM-OECs' viability, which we have accumulate some experoence. And we will conduct Magnetic Resonance Diffusion Tensor Imaging (MR-DTI) to assess the spinal cord, which is very of sensitivity and accuracy in the living rats. Moreover, other measurments include cell couting, cell MTT, cell identification, serum diagnosis (inflammatory factor), active factors test (TGF-α, NT-3, BDNF, NGF and so on), spinal cord Oil dyeing and Western-blot (NogoA, Semaphorin3A, MAG, OMgP, NgR and RhoA). When the whole research finish we could clarify the effect and mechanism of GBCs/HBCs/OM-MSCs from human olfactory mucosa on OM-OECs transplantation treatment for SCI, which will offer a new approach to gain the seed cells for the transplantation treatment of SCI.

本项目拟先用NT-3/TGF-α培养提纯、后用FACS技术分选人嗅粘膜OECs，同时直接分选嗅粘膜另外3种主要细胞（球状/水平基底细胞GBCs、HBCs和间充质干细胞MSCs），进而将GBCs、HBCs和OM-OECs分别与OM-OECs进行体外共培养，检测培养液中活性因子种类和含量并定量测定OM-OECs的NT-3基因表达以评价细胞活性，以揭示此3种细胞对OM-OECs活性的协同作用强度和机制，进而以人嗅球OECs做对照进行大鼠脊髓损伤体内验证，明确4种嗅粘膜来源细胞治疗SCI的修复作用强度。我们初步发现NT-3有助于OM-OECs的提纯，OM-MSCs与OM-OECs共培养具有协同促进作用，将用磁共振弥散张量成像技术对脊髓进行客观评价，并测其他细胞、生化和组织学指标。通过本研究可揭示人嗅粘膜GBCs、HBCs和MSCs在OECs治疗SCI中的协同作用机制，以提供移植用细胞新的来源选择。

本项目研究着眼于目前脊髓损伤修复细胞移植治疗研究中心被最早应用于临床且治疗效果最确切的嗅鞘细胞为研究对象，从嗅粘膜嗅鞘细胞的培养出发，培养出了高纯度的嗅鞘细胞，并研究了不同浓度及不同种类血清对体外培养大鼠嗅鞘细胞的影响。研究发现体外培养中，不同种类的血清均可以培养出纯度较高、用于脊髓损伤后移植的嗅鞘细胞；在血清浓度一致的情况下，15%胎牛血清比大鼠血清更利于嗅鞘细胞增殖；持续使用含有胎牛血清培养基的嗅鞘细胞，其数量、纯度以及分泌NT-3的含量均优于其他方法培养的嗅鞘细胞。研究中发现嗅鞘细胞对于脊髓损伤修复作用，其中一部分可能是通过Reelin产生的；然而在体内实验中，嗅鞘细胞的移植并没有使Reelin的表达出现明显的上升，因此说明嗅鞘细胞在损伤局部的修复功能可以通过Reelin起作用、但是并不影响局部Reelin本身的表达。在研究中还发现嗅鞘细胞上清液也可以发挥较好的神经修复作用，所以继续开展了嗅鞘细胞及其上清液对大鼠脊髓损伤修复作用及DTI在其中应用的研究。研究发现差速贴壁后培养至9d时的OECs细胞活性及纯度最佳；DTI扫描是评价OECs移植治疗脊髓损伤疗效的敏感指标，且DTI参数与OECs移植后的BBB评分的密切相关；持续腹腔注射OECs上清液能够促进SCI后运动功能恢复，增强损伤局部细胞自噬。为深入研究嗅鞘细胞与间充质干细胞的协同作用，本研究以间充质干细胞中的Muse细胞为切入点，研究了人脐带来源Muse干细胞的定量分析、筛选纯化及其移植治疗大鼠脊髓损伤的疗效。结果显示人脐带是新型Muse干细胞的良好来源；环磷酰胺可用于从WJ-MSCs中提纯Muse细胞，最佳方案是20mM浓度干预24h；免疫磁珠筛选-细胞培养-再次免疫磁珠筛选方法可以得到大量高纯度的Muse细胞；验证了Muse细胞移植治疗大鼠亚急性脊髓损伤是安全有效的。本项目从嗅鞘细胞来源的问题出发，解决了培养中的技术问题，提出了新的治疗靶点，发现了新的治疗途径，提出了适宜的纯化方法，为临床治疗脊髓损伤从多方面提供了支撑。