Development of novel therapeutic means to regenerating spinal cord injury using microglia/macrophages

小胶质细胞被认为是大脑中唯一起源于中胚层的细胞群，尽管它们的作用尚不完全清楚。本研究表明，大鼠原代小胶质细胞表达巢蛋白、A2B5和O4抗原，这些抗原是少突胶质前体细胞的标记物。基于这些发现，我们研究了是小胶质细胞生成神经元还是大胶质细胞。纯化的小胶质细胞在10%胎牛血清中培养3 d，然后在70%血清中培养2 d。在两步培养过程中，小胶质细胞变得高度增殖，并强烈表达DNA结合抑制剂（Id）基因，表明细胞去分化。去分化细胞也表达促进分化为神经元或大胶质细胞的转录因子。将去分化细胞转移到无血清培养基中，大量细胞迅速转化为长过程细胞，表达微管相关蛋白2、突触素1、神经丝蛋白、胶质纤维酸性蛋白或半乳糖脑苷。当通过乙酰化低密度脂蛋白（LDL）受体或吞噬依赖机制对小胶质细胞进行荧光标记时，可以观察到携带荧光的神经元、星形胶质细胞或少突胶质细胞。神经干细胞聚集的神经球表达武藏1和表皮生长因子受体，而小胶质细胞不表达。这些结果表明，小胶质细胞作为多潜能干细胞在产生神经元、星形胶质细胞或少突胶质细胞方面具有新的作用。这种被称为前小胶质母细胞（promicroglioblasts, promgb）的多能MG形成细胞聚集体，在转移到无血清培养基后不久产生具有神经外胚层表型的细胞。免疫组化结果显示，新生大鼠脑组织中有少量表达NG2硫酸软骨素蛋白多糖（NG2）的MG。来自新生儿大脑的原代培养物含有NG2^+ MG，这似乎是NG2^+ ProMGB聚集体的来源。聚集体为MG标记物^+/NG2^+/GFAP^+/NCAM^+/S^-100b^-，具有碱性磷酸酶活性。在成熟大鼠脑刺伤处观察到明显的NG2^+ MG积累。伤口中累积的NG2 + MG逐渐减少，但细胞持续存在至损伤后150天。此外，GFAP在创面周围的免疫反应性明显增强。经胰蛋白酶- edta分离的创面NG2^+ MG在70%血清添加培养基中形成NG2^+聚集体，在无血清培养基中转化为具有神经外胚层表型的细胞。虽然从成熟的大脑中分离活神经元是困难的，但在体外，刺伤细胞很容易产生承载过程的b-微管蛋白III^+细胞。这些数据表明，正常发育或病理大脑中的NG2^+ MG参与了大脑的发生或再生。少

Microglia are considered the only cell population of mesodermal origin in the brain, although their role is not fully understood. The present study demonstrated that rat primary microglial cells expressed nestin, A2B5, and O4 antigens, which are markers for oligodendrocyte precursor cells. Based on these findings, we investigated whether microglial cells generated neurons or macroglial cells. Purified microglial cells were cultured in the presence of 10% fetal bovine serum for 3 d, followed by culture in the presence of 70% serum for 2 d. During the two-step culture, microglial cells became highly proliferative and strongly expressed inhibitor of DNA binding (Id) genes, indicative of dedifferentiation of the cells. The dedifferentiated cells also expressed transcription factors that promote differentiation into neurons or macroglial cells. When the dedifferentiated cells were transferred into serum-free medium on poly-L-lysine-coated substrate, a substantial number of the cells rapidly … More turned into long process-bearing cells, which expressed microtubule-associated protein 2, synapsin I, neurofilament proteins, glial fibrillary acidic protein, or galactocerebroside. When microglial cells were fluorescently labelled through acetylated low-density lipoprotein (LDL) receptors or by a phagocytosis-dependent mechanism, fluorescence-bearing neurons, astrocytes, or oligodendrocytes were observed. Neurospheres, aggregates of neural stem cells, expressed Musashi 1 and epidermal growth factor receptor, but the microglia-derived cells did not. These results suggest a novel role of microglia as multipotential stem cells to give rise to neurons, astrocytes, or oligodendrocytes.Such multipotent MG called promicroglioblasts (ProMGBs) formed cell aggregates, which generated cells with neuroectodermal phenotypes shortly after their transfer into serum-free medium. As revealed by immunohistochemistry, there were a few MG expressing NG2 chondroitin sulfate proteoglycan (NG2) in the neonatal rat brain. Primary culture from the neonatal brain contained NG2^+ MG, which appeared to be the source of NG2^+ ProMGB aggregates. The aggregates were MG marker^+/NG2^+/GFAP^+/NCAM^+/S^-100b^- and had alkaline phosphatase activity. The marked accumulation of NG2^+ MG was observed close to stab wounds made in the mature rat brain. The accumulated NG2^+ MG in the wound gradually decreased in number, but the cells persisted up to 150 days postlesioning. In addition, GFAP immunoreactivity increased markedly around the wound. The NG2^+ MG in the wounds separated with trypsin-EDTA formed NG2^+ aggregates in 70% serum-supplemented medium and then transformed into cells with neuroectodermal phenotypes in serum-free medium. Although it is difficult to separate viable neurons from mature brains, cells from stab wounds generated process-bearing b-tubulin III^+ cells in vitro easily. These data suggest that NG2^+ MG in normal developing or pathologic brains are involved in the genesis or regeneration of the brain. Less