细胞协同/互补效应对脊髓损伤的治疗与再生微环境调节作用

At present, therapy based on the cells' synergic or complementary effects for spinal cord injury is one of focus issues. Both olfactory ensheathing cells (OECs) and Schwann cells (SCs) have a cell-mediated therapic effects for spinal cord injury. Previously it has been confirmed that OECs are more potential for promoting nerve to regenerate, with a ability of active migration, more extensive distritution after transplantion to the injured spinal cord. While SCs are more potential for remyelination, but migrate and distribute lessly. Recently, our study showed that OECs and SCs have a synergic or complementary effects in repairing sciatic nerve defect. We also revealed recently that OECs can protect intact tissue and reduce injured area in the contused spinal cord, which may be related to the neuro-immunology and inflammation. In this project, after contusion model of rat spinal cord being made, which is close to human injury model, suspension of OECs mixed SCs will be transplanted into the contused cord. Then the donor cells' survival, migration, distribution in vivo and their potential for inhibitating second injury, reducing formation of glial scar, promoting nerve regeneration, and remyelination will be qualitatively and quantitatively observed for determining synergic/complementary effects in the recovery of structure and function of SCI. Meanwhile, expression of typical inhibitory proteins such as Nogo-A and CSPGs, neurotrophines such as BDNF and GDNF, macrophages and cytokines will be assessed for approaching synergic or complementary effects on mediation of micro-environment. The aim of the project is to provide exprimental evidences for the cell-mediated therapy of SCI in clinic.

利用细胞协同/互补效应治疗脊髓损伤是当前的研究热点之一。OECs促脊髓神经纤维再生的能力更强，迁移活跃，分布广泛；SCs对脊髓再生神经纤维有更强的成髓鞘作用，但迁移性小，分布范围窄。我们最近证明，两者在周围神经损伤修复中具有协同/互补作用。也证实OECs可以保护损伤脊髓的健康组织，缩小损伤范围，推测和局部免疫反应有关。为此，本项目在制作接近人类的脊髓挫伤模型后，混合移植两种细胞，观察它们在挫伤脊髓内的存活、迁移和分布，对继发性损伤和胶质瘢痕形成的抑制作用，对下行神经纤维的促再生作用，对再生神经纤维的成髓鞘作用，对挫伤脊髓功能的恢复作用，以确定两者协同/互补效应在治疗挫伤脊髓结构和功能中的作用；并通过观察Nogo-A和CSPGs等抑制蛋白、BDNF和GDNF等神经营养因子、巨噬细胞和相关细胞因子的表达变化，试图探讨协同/互补效应对局部微环境的调节作用，从而为临床治疗脊髓损伤提供实验学依据。

利用细胞协同/互补效应治疗脊髓损伤是当前的研究热点之一。OECs和SCs均是对脊髓再生具有重要促进作用的种子细胞。本项目在制作接近人类的脊髓挫伤模型后，混合移植两种细胞，观察它们对挫伤脊髓的结构和功能是否具有协同/互补作用；并探讨协同/互补效应对局部微环境的调节作用，从而为临床治疗脊髓损伤提供实验学依据。. 在项目研究过程中，我们发现联合移植OECs和SCs可显著促进移植细胞细胞在脊髓内的迁移，其分布面积，迁移距离均显著优于单一细胞移植组。联合移植OECs和SCs组的GFAP表达也显著低于单一细胞或DMEM对照组，说明两种细胞在抑制胶质瘢痕增生的作用上具有协同效应。OECs和SCs联合移植组在促进挫伤脊髓组织结构（空洞形成）和运动功能恢复方面（BBB评分、电生理）都有显著的协同效应，其作用显著优于单一细胞或DMEM组。. 这些协同效应是否与炎症微环境的调节作用相关呢？因此，本项目组更深入的研究了各组挫伤脊髓局部巨噬细胞亚型的分布，以及各种炎症因子的表达情况。我们发现联合移植OECs和SCs组的巨噬细胞向M2亚型极化的数量增多，而向M1亚型极化的数量减少。因此，我们进一步验证了脊髓内促进巨噬细胞不同极化方向的多种趋化因子和刺激因子的表达量，联合移植组的趋化因子CCL2和CCL3均表达降低，IL-4（M2巨噬细胞刺激因子）表达增加，IFN-r（M1巨噬细胞刺激因子）表达降低。这些因子的表达变化也进一步验证了联合移植两种细胞可协同/互补促进巨噬细胞向M2亚型极化，有利于脊髓损伤的修复。联合移植组IL-6，TNF-a，的表达显著低于其他各组，IL-10，IL-13的表达显著高于其他各组。说明两种细胞移植可协同促进抗炎因子的表达，抑制促炎因子的表达，对炎症微环境有调节作用。这些研究结果为细胞移植治疗脊髓损伤提供了重要的实验学依据。