Jonakait G M, Luskin M B, Wei R, Tian X F, Ni L
Department of Biological Sciences, Rutgers University, Newark, New Jersey, 07102, USA.
Dev Biol. 1996 Jul 10;177(1):85-95. doi: 10.1006/dbio.1996.0147.
In earlier studies we found that treatment with interferon-gamma (IFN-gamma) produced an 8- to 11-fold increase in choline acetyltransferase (ChAT) in cultured cells taken from Embryonic Day 16 (E16) septal nuclei with adjacent basal forebrain (SN/BF). Since younger cultures responded even more profoundly to IFN treatment, we have tested the possibility that the action of IFN (or its intermediate; see below) is to prompt the cholinergic differentiation of neuronal precursors. SN/BF cultures of various ages were labeled with a retrovirus engineered to express beta-galactosidase (Lac-Z), and ChAT-positive descendants of the retrovirally labeled precursors were counted. IFN-gamma treatment of cultures caused as much as an 8.8-fold increase in the proportion of ChAT-positive cells present in Lac-Z-positive clones, suggesting that IFN promoted cholinergic differentiation in precursor populations. By contrast, bFGF increased clone size but did not change the proportion of ChAT-positive cells. NGF affected neither. Only ameboid microglia present in the cultures responded to IFN with characteristic nuclear translocation of the signal transducing molecule p91, suggesting that a microglial-derived molecule may mediate the action of IFN. Consistent with this hypothesis, conditioned media from cultures of enriched, activated microglia also increased ChAT activity in a dose-dependent fashion. Conditioned media from an unstimulated macrophage/monocyte cell line (RAW 264.7) also proved extremely efficacious in raising ChAT activity. In addition, conditioned media from both activated microglia and RAW 264.7 cells increased the proportion of ChAT-positive cells in retrovirally labeled clones to the same extent as IFN itself, suggesting the possibility that they contain the molecule(s) that mediates the action of IFN. Preliminary characterization of this molecule suggests that it is a very stable and large protein. Together these data suggest that a molecule promoting cholinergic differentiation is produced by activated microglia and other macrophage-like cells. The identity of this molecule and its precise role in normal development await its further purification.
在早期研究中，我们发现用γ-干扰素（IFN-γ）处理取自胚胎第16天（E16）隔核与相邻基底前脑（SN/BF）的培养细胞时，胆碱乙酰转移酶（ChAT）增加了8至11倍。由于较年轻的培养物对IFN处理反应更为强烈，我们测试了IFN（或其中间体；见下文）的作用是否是促使神经元前体向胆碱能分化的可能性。用经基因工程改造以表达β-半乳糖苷酶（Lac-Z）的逆转录病毒标记不同年龄的SN/BF培养物，并对逆转录病毒标记前体的ChAT阳性后代进行计数。用IFN-γ处理培养物导致Lac-Z阳性克隆中ChAT阳性细胞的比例增加多达8.8倍，这表明IFN促进了前体细胞群中的胆碱能分化。相比之下，碱性成纤维细胞生长因子（bFGF）增加了克隆大小，但没有改变ChAT阳性细胞的比例。神经生长因子（NGF）对两者均无影响。培养物中仅存在的阿米巴样小胶质细胞对IFN有反应，信号转导分子p91发生特征性核转位，这表明小胶质细胞衍生的分子可能介导IFN的作用。与此假设一致，来自富集的、活化的小胶质细胞培养物的条件培养基也以剂量依赖性方式增加了ChAT活性。来自未刺激的巨噬细胞/单核细胞系（RAW 264.7）的条件培养基在提高ChAT活性方面也被证明极其有效。此外，来自活化小胶质细胞和RAW 264.7细胞的条件培养基将逆转录病毒标记克隆中ChAT阳性细胞的比例增加到与IFN本身相同的程度，这表明它们可能含有介导IFN作用的分子。对该分子的初步表征表明它是一种非常稳定的大蛋白。这些数据共同表明，活化的小胶质细胞和其他巨噬细胞样细胞产生一种促进胆碱能分化的分子。该分子的身份及其在正常发育中的精确作用有待进一步纯化。
