PD-L1对小鼠脊髓损伤后星形胶质细胞反应性增生和胶质瘢痕形成的负性调控机制研究

Reactive astrogliosis (RA) evolves as time elapses and finally results in the formation of glial scar (GA) by a series of positive feedback after spinal cord injury (SCI), which inhibits the axon regeneration. Currently, however, whether the negative signaling pathway is involved in the controlling of the process of RA and GC remains unclear. Our preliminary observations have shown that PD-L1, as a negative regulatory molecular, shows upregulated expression in astrocytes after spinal cord injury. The degree of GA formation in PD-L1high mice is less than that in wild type mice. While the ability of migration in cultured astrocytes from PD-L1high mice is weaker than that from wild type mice. These evidences strongly suggest that the involvement of PD-L1 in the regulation of RA. We hypothesize that activated astrocytes may negatively control the process of RA and GC by the upregulating expression of PD-L1 and binding to PD-1 or/and CD80 to promote the recovery of spinal cord injury. In this project, we shall perform in vivo and in vitro experiments, using many kinds of gene mice, to testify roles of PD-L1 in the regulation of RA and GC in the model of spinal cord injury. It is expected that the underlying mechanism for negatively controlling RA and GC and new targets for the repair of SCI will be elucidated.

脊髓损伤（SCI）后星形胶质细胞反应性增生（RA），并通过正反馈环路加重反应形成胶质瘢痕(GC)，严重抑制轴突再生。但目前尚不清楚是否存在内源性负调控信号通路调控RA和GC的发生与发展。PD-L1是一种负调控分子，前期SCI的研究中发现：PD-L1在星形胶质细胞中表达显著上调；高表达PD-L1小鼠SCI后的GC明显轻于野生型小鼠，同时细胞迁移能力明显弱于敲除PD-L1的细胞。这些结果强烈提示：PD-L1可能参与RA的调控。我们推测：SCI后，活化的星形胶质细胞可能通过其表面高表达PD-L1以及与PD-1和/或CD80的相互作用，传递负调控信号，内源性的调控RA及GC的形成，促进SCI的修复。本课题拟采用多种基因修饰小鼠，通过在体和离体实验，阐明PD-L1在SCI导致的RA及GC形成过程中的作用机制及可能的信号通路，探讨RA和GC的内源性负调控机制，为今后SCI的治疗提供新靶点及理论依据。

脊髓损伤（SCI）后星形胶质细胞反应性增生（RA），并通过正反馈环路形成胶质瘢痕(GC)，严重抑制轴突再生。本项目计划研究调控RA和GC发生与发展的内源性负调控信号通路。PD-L1作为一个重要的免疫抑制信号分子，通过传递负性调控信号维持免疫耐受和细胞稳态。因此项目按计划执行，以PD-L1分子作为切入点，采用PD-L1-/-（PD-L1 基因缺失）和TgPD-L1 转基因（PD-L1 高表达）小鼠，通过在体和离体实验，探讨PD-L1在SCI导致的RA及GC形成过程中的作用机制及可能的信号通路。结果发现：1）PD-L1在体外培养的AS中弱表达，给予LPS+INFγ刺激后，PD-L1表达显著升高。来源PD-L1-/-小鼠大脑皮层的AS原代培养细胞形态发生改变，表现类似上皮细胞，内部骨架蛋白聚合现象较野生型组明显，应力纤维数量明显多于野生型组，排列趋向一致。2）对两种转基因小鼠的AS进行Q-PCR检测，发现PD-L1敲除后，与细胞骨架结构相关GFAP、Vimentin、S100β、Bystin mRNA表达较野生型AS相比均明显下调；与增殖相关Ki67、cdk、ccnd1、ccnb1和ccnb2也发生表达下调。给予LPS+INFγ刺激后，野生型和敲除鼠的AS均被活化。PD-L1敲除小鼠AS的增殖水平低于野生型AS，炎性条件会进一步抑制PD-L1敲除小鼠AS的增殖。3）AS细胞进行划伤实验，活细胞工作站连续检测发现PD-L1敲除的AS迁移速度快于野生型。WB检测发现PD-L1敲除AS中E-cadherin下调，N-cadherin、paxinllin和β-catenin表达上调。给予LPS+INFγ刺激后，两者的表达差异会更加显著。4）野生型小鼠SCI后，PD-L1的表达水平随损伤时间逐渐增加，除了在活化的巨噬细胞/小胶质细胞表达外，也主要表达在增生活化的AS中。PD-L1敲除小鼠脊髓组织中，GFAP、iNOS表达上调，同时与细胞外基质和迁移相关的蛋白如E-cad、N-cad也发生显著改变，5）动物SCI后，PD-L1敲除小鼠BMS评分明显好于野生型。6）利用U251细胞系初步探讨，认为PD-L1可能通过TGF-smad信号途径调控RA和GC的形成。综上所述，该课题明确PD-L1可负性调控脊髓损伤后RA和GC的形成，促进SCI的修复，这为今后SCI的治疗提供新靶点及理论依据。