甲酰基肽受体调节小胶质细胞表型转换和趋化在脊髓损伤中的作用与机制

Impaired function recovery from spinal cord injury (SCI) is mainly attributed to deteriorate immune microenvironment at the injurious site which is mainly based on infiltrating immune cells and resident microglia. Microglias are the main effector cells post SCI, which have double-edged effects for neural tissue based on the number of infiltration and polarization of M1 M2 phenotype. M1 microglias accelerate neural dysfunction, while M2 microglias protect neural tissue from secondary injury. Formylpeptide receptors (FPRs) are promiscuously but mainly expressed in myeloid cells and primarily responsible for migration of phagocytes through ligation with spcific ligands (agonists and antagonists), such as microbial products, endogenous agonists and synthetic peptides. Our previous study uncovered the defect of microglial infiltration with higher M2 proportion in the lesion site of SCI correlated with better function recovery in FPR knockout mice, compared with wild type (WT) mice. We hypothesize that knockout of FPRs induces reduced accumulation of microglia and polarizes microglias to protective M2 phenotype to regulate the microenvironment and function recovery from contused SCI. To confirm this hypothesis, the expression of FPR agonists at injurious sites and FPRs in microglias will be detected with N9 cell line and primary microglia from WT and FPR-deficient, FPR-transgenic mice in vitro; in addition, the function recovery from contused SCI will be assayed, the kinetics and phenotypes of infiltrating microglias will be determined with WT, FPR knockout and FPR-transgenic mice. The melecular mechanistic basis will be exploited. The study will strengthen the understanding of mechnisms and shed light on therapy strategy of SCI.

脊髓损伤（SCI）局部不良炎性微环境是限制神经功能恢复的重要因素。小胶质细胞是中枢神经系统（CNS）的主要炎性细胞，据其表型不同而对神经功能恢复起到“双刃剑”的作用：M1表型小胶质细胞起损伤作用，M2表型起保护作用。预实验显示：甲酰基肽受体（FPRs）基因敲除小鼠SCI局部存在FPRs配体，小胶质细胞在损伤区聚集减少，但M2表型比例增加，神经功能恢复优于野生型小鼠，提示SCI局部FPRs在小胶质细胞表型转换与趋化、调控微环境中起重要作用。本课题设想：SCI局部的内源性FPR配体与小胶质细胞表达的FPRs结合而趋化后者在损伤区聚集，如敲除FPR基因则小胶质细胞趋化减少，M2表型比例增加。本课题拟使用FPR基因敲除、FPR转基因和嵌合体小鼠制作SCI模型，体内外检测小胶质细胞表型转换、浸润规律、信号途径及其对神经功能恢复的影响。研究将深化对SCI机制的认识并为治疗SCI提供新思路。

脊髓损伤（SCI）发病率逐年增高，而致残率高、死亡率低，大量患者需终生照护，极大地增加了医疗负担，因此加强SCI研究意义重大。本课题围绕SCI修复过程中起重要作用的小胶质细胞和神经干细胞（NSCs）展开研究，探讨甲酰基肽受体（FPR）对其作用，尤其是迁移和分化方面的作用及机制，为SCI的治疗提供新的思路。另外，医源性SCI是椎管内肿瘤切除术常见的并发症之一，常合并脊柱稳定性破坏，而重建脊柱稳定性会导致活动功能的丧失，因此我们还在临床工作中采用后方韧带复合体（PLC）回植重建术后脊柱稳定性并观察其效果。基于此，我们取得下述成果：1.明确了小胶质细胞系BV-2细胞能够表达FPRs，且主要位于细胞膜表面。2.FPRs对小胶质细胞具有特异性的趋化作用。FPR1激动剂fMLF趋化小胶质细胞迁移，而能被FPR1特异性阻断剂Boc-MLF抑制，该作用通过上调信号分子ERK的磷酸化而实现。3.首次发现NSCs能够表达功能性FPR1和FPR2。采用多种技术在基因、蛋白水平证实NSCs表达FPR1和FPR2，并将其定位于细胞质和细胞膜。4.FPR1和FPR2能够特异性趋化NSCs迁移。除体外形态学观察和transwell法证实FPR1和FPR2对NSC迁移的特异性外，还采用两种体内模型证实FPRs能够显著趋化移植或内源性NSCs沿激动剂浓度梯度向激动剂注射区迁移，而FPRs特异性阻断剂能够特异性阻断该作用，充分证明了在体内FPRs能够特异性地趋化NSCs向FPR激动剂部位募集，从而使FPRs成为潜在的治疗靶点。5.FPRs能够强化NSCs向功能性神经元分化。研究发现FPR1激动剂fMLF、FPR2激动剂MMK-1促进NSCs向神经元分化，而对NSCs向星形胶质细胞和少突胶质细胞分化无明显的作用。6.课题同时联合使用神经营养因子-3（NT-3）以增强NSCs支持性微环境，结果显示NT-3通过PI3K/AKT信号途径显著上调鼠脑源性NSC中SOX2表达，并明显促进NSC向神经元方向分化。7.后方韧带复合体回植能够有效重建椎管内肿瘤切除术后脊柱稳定性而保留脊柱功能性运动节段，为神经功能恢复创造良好的条件。.综合上述，我们证实了FPRs在小胶质细胞迁移中起重要作用；首次发现NSCs表达功能性FPRs，并在其迁移、分化中起重要作用，为SCI治疗提供了新的策略。