CLOSING THE PHENOTYPIC GAP BETWEEN TRANSFORMED AND UNTRANSFORMED NEURONS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 中枢神经系统(CNS)神经元功能障碍的研究常常受到原代神经元体外培养失败的限制。神经细胞系可以替代原代细胞，但它们经常错误地反映正常情况。我们假设，三维(3D)细胞培养系统将推动转化的神经元的表型更接近于未转化的细胞，就像在非神经元细胞系中所展示的那样。在我们的研究中，比较3D和2D培养，神经元SH-SY5Y(SY)细胞经历了明显的形态变化，细胞分裂率显著下降。与标准2D条件相比，3D培养中原癌基因N-myc和RNA结合蛋白HUD的表达降低。我们观察到，在3D培养中，抗凋亡蛋白Bcl2的表达下降，而促凋亡蛋白Bax和Bak的表达增加。此外，thapsigargin(TG)诱导的3D细胞的凋亡增强。微阵列分析显示两种培养类型的细胞中700多个基因的基因表达水平显著不同，并表明G1/S细胞周期进程的改变导致3D培养的SY细胞倍增率降低。这些结果表明，3D培养方法缩小了神经元细胞系和原代神经元之间的表型差距。所得到的细胞可容易地用于神经元发病机制的体外研究。一篇包含这些结果的论文发表在《神经科学方法杂志》上。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Studies of neuronal dysfunction in the central nervous system (CNS) are frequently limited by the failure of primary neurons to propagate in vitro. Neuronal cell lines can be substituted for primary cells but they often misrepresent normal conditions. We hypothesized that a three-dimensional (3D) cell culture system would drive the phenotype of transformed neurons closer to that of untransformed cells, as has been demonstrated in non-neuronal cell lines. In our studies comparing 3D versus two-dimensional (2D) culture, neuronal SH-SY5Y (SY) cells underwent distinct morphological changes combined with a significant drop in their rate of cell division. Expression of the proto-oncogene N-myc and the RNA-binding protein HuD was decreased in 3D culture as compared to standard 2D conditions. We observed a decline in the anti-apoptotic protein Bcl-2 in 3D culture, coupled with increased expression of the pro-apoptotic proteins Bax and Bak. Moreover, thapsigargin (TG)-induced apoptosis was enhanced in the 3D cells. Microarray analysis demonstrated significantly differing mRNA levels for over 700 genes in the cells of the two culture types, and indicated that alterations in the G1/S cell-cycle progression contributed to the diminished doubling rate in the 3D-cultured SY cells. These results demonstrate that a 3D culture approach narrows the phenotypic gap between neuronal cell lines and primary neurons. The resulting cells may readily be used for in vitro research of neuronal pathogenesis. A paper including these results was published in the Journal of Neuroscience Methods.