PURIFICATION AND CHARACTERZATION OF GLIA-DERIVED NEUROPROTECTIVE FACTORS

我们在这个项目中发现了以下神经保护作用：1)小胶质细胞的神经保护作用。我们用一氧化氮(NO)供体化合物硝普钠(SNP)孵育大鼠大脑皮层原代神经元，发现神经元死亡。利用该模型，我们研究了小胶质细胞对神经元存活的影响。结果表明，与神经元共培养的小胶质细胞可完全阻止NO诱导的神经细胞凋亡。由于小胶质细胞条件培养液也在很大程度上抑制了神经元的凋亡，因此小胶质细胞来源的抗凋亡因子可能是一种可溶性因子(S)。此外，条件培养液在暴露于NO后使用时也有效。在小胶质细胞产生的各种已知细胞因子和生长因子中，只有肿瘤坏死因子a显著拯救了神经元免受NO诱导的损伤。2)星形胶质细胞的神经保护作用。我们研究了星形胶质细胞是否可以从活性氧(ROS)诱导的原代大脑皮层神经元的凋亡中拯救出来。在神经元培养板中加入硝普钠、3-吗啉琥珀酸亚胺(SIN-1)或FeSO4与抗坏血酸的混合物，加入NO、超氧阴离子或羟基自由基。当星形胶质细胞与神经元共同培养时，ROS诱导的神经元死亡被完全阻止。然而，脑膜成纤维细胞不能挽救神经元。用星形胶质细胞来源的细胞外基质(ECM)培养神经元，可以最大限度地防止神经元的凋亡。星形胶质细胞来源的ECM含有层粘连蛋白和纤维连接蛋白，纯化的层粘连蛋白和纤维连接蛋白显著保护神经元免受氧化应激。

We have found the followings regarding the neuroprotective effects in this project.1) Neuroprotective effects of microglial cells. We found that cerebrocortical primary neurons died by apoptosis by incubating cells with sodium nitroprusside (SNP), a nitric oxide (NO)-donor compound, By the use of this apoptosis model, we investigated the effect of microglial cells on neuronal survival. As a result, the NO-induced neuronal apoptosis was completely prevented by microglial cells that were cocultured with neurons. Since the neuronal apoptosis was also largely inhibited by microglial conditioned medium, microglia-derived anti-apoptotic factor may be a soluble-factor(s). Furthermore, the conditioned medium was effective when it was applied after exposure to NO. Among a variety of known cytokines and growth factors that are produced by microglial cells, only tumor necrosis factor a significantly rescued neurons against NO-induced damage. 2) Neuroprotective effects of astrocytes. We investigated to determine whether astrocytes could rescue primary cerebrocortical neurons from reactive oxygen species (ROS)-induced apoptosis. By adding SNP, 3-morpholinosydononimine (SIN-1) or mixture of FeSO4 and ascorbic acid, NO, superoxide anion or hydroxyl radicals were introduced into culture plates of neurons. When astrocytes were cocultured with neurons, ROS-induced neuronal death was completely prevented. However, meningeal fibroblasts could not rescue neurons. When neurons were cultured on astrocytes-derived extracellular matrix (ECM), neuronal apoptosis was mostly prevented. The astrocyte-derived ECM contained laminin and fibronectin, and purified laminin and fibronectin significantly protected neurons against oxidative stress.