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In vitro effects of fetal rat cerebrospinal fluid on viability and neuronal differentiation of PC12 cells

机译：胎鼠脑脊髓液对PC12细胞活力和神经元分化的体外影响

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Background Fetal cerebrospinal fluid (CSF) contains many neurotrophic and growth factors and has been shown to be capable of supporting viability, proliferation and differentiation of primary cortical progenitor cells. Rat pheochromocytoma PC12 cells have been widely used as an in vitro model of neuronal differentiation since they differentiate into sympathetic neuron-like cells in response to growth factors. This study aimed to establish whether PC12 cells were responsive to fetal CSF and therefore whether they might be used to investigate CSF physiology in a stable cell line lacking the time-specific response patterns of primary cells previously described. Methods In vitro assays of viability, proliferation and differentiation were carried out after incubation of PC12 cells in media with and without addition of fetal rat CSF. An MTT tetrazolium assay was used to assess cell viability and/or cell proliferation. Expression of neural differentiation markers (MAP-2 and β-III tubulin) was determined by immunocytochemistry. Formation and growth of neurites was measured by image analysis. Results PC12 cells differentiate into neuronal cell types when exposed to bFGF. Viability and cell proliferation of PC12 cells cultured in CSF-supplemented medium from E18 rat fetuses were significantly elevated relative to the control group. Neuronal-like outgrowths from cells appeared following the application of bFGF or CSF from E17 and E19 fetuses but not E18 or E20 CSF. Beta-III tubulin was expressed in PC12 cells cultured in any media except that supplemented with E18 CSF. MAP-2 expression was found in control cultures and in those with E17 and E19 CSF. MAP2 was located in neurites except in E17 CSF when the whole cell was positive. Conclusions Fetal rat CSF supports viability and stimulates proliferation and neurogenic differentiation of PC12 cells in an age-dependent way, suggesting that CSF composition changes with age. This feature may be important in vivo for the promotion of normal brain development. There were significant differences in the effects on PC12 cells compared to primary cortical cells. This suggests there is an interaction in vivo between developmental stage of cells and the composition of CSF. The data presented here support an important, perhaps driving role for CSF composition, specifically neurotrophic factors, in neuronal survival, proliferation and differentiation. The effects of CSF on PC12 cells can thus be used to further investigate the role of CSF in driving development without the confounding issues of using primary cells.
机译：背景技术胎儿脑脊髓液（CSF）包含许多神经营养和生长因子，并已被证明能够支持原代皮质祖细胞的活力，增殖和分化。大鼠嗜铬细胞瘤PC12细胞已被广泛用作神经元分化的体外模型，因为它们响应生长因子而分化为交感神经元样细胞。这项研究旨在确定PC12细胞是否对胎儿CSF有反应，因此是否可以将它们用于在缺乏前述原代细胞时间特异性反应模式的稳定细胞系中研究CSF生理学。方法在添加和不添加胎鼠脑脊液的培养基中孵育PC12细胞后，进行体外生存力，增殖和分化测定。使用MTT四唑鎓测定法评估细胞活力和/或细胞增殖。通过免疫细胞化学测定神经分化标志物（MAP-2和β-III微管蛋白）的表达。通过图像分析测量神经突的形成和生长。结果当暴露于bFGF时，PC12细胞分化为神经元细胞类型。相对于对照组，在补充了E18大鼠胎儿CSF的培养基中培养的PC12细胞的活力和细胞增殖显着提高。应用来自E17和E19胎儿的bFGF或CSF，但未出现E18或E20 CSF的细胞出现神经元样生长。 Beta-III微管蛋白在除添加E18 CSF以外的任何培养基中培养的PC12细胞中表达。在对照培养物中以及在具有E17和E19 CSF的培养物中发现了MAP-2表达。当整个细胞为阳性时，MAP2位于神经突中，除了E17 CSF中。结论胎鼠脑脊液支持生命，并以年龄依赖性方式刺激PC12细胞的增殖和神经原性分化，表明脑脊液成分随年龄而变化。该特征在体内对于促进正常大脑发育可能是重要的。与原代皮层细胞相比，对PC12细胞的作用存在显着差异。这表明在细胞的发育阶段和CSF的组成之间存在体内相互作用。此处提供的数据支持脑脊液成分（特别是神经营养因子）在神经元存活，增殖和分化中的重要作用，也许是驱动作用。因此，CSF对PC12细胞的作用可用于进一步研究CSF在驱动发育中的作用，而不会引起使用原代细胞的混淆问题。

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