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(3-D)细胞培养系统将使转化的神经元的表型更接近于未转化的细胞。在我们的研究中，比较三维和二维培养，神经元SH-SY5Y(SY)细胞发生了明显的形态变化，细胞分裂率显著下降。与标准的二维培养相比，三维培养中原癌基因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3-D细胞的凋亡增强。微阵列分析显示，在每种培养类型的细胞中，700多个基因的mRNA水平存在显著差异。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。

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 3-dimensional (3-D) cell culture system would drive the phenotype of transformed neurons closer to that of untransformed cells. In our studies comparing 3-D versus 2-dimensional (2-D) 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 3-D culture as compared to standard 2-D conditions. We observed a decline in the anti-apoptotic protein Bcl-2 in 3-D culture, coupled with increased expression of the pro-apoptotic proteins Bax and Bak. Moreover, thapsigargin (TG)-induced apoptosis was enhanced in the 3-D cells. Microarray analysis demonstrated significantly differing mRNA levels for over 700 genes in the cells of each culture type. These results indicate that a 3-D 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.