Role of the human transcription factor T in chordoma growth

在这个项目的第二年，我们执行了必要的材料转移协议，以获得已公布的脊索瘤细胞系，包括新的脊索瘤细胞系UCH-2。我们已经测试了两个已知的脊索瘤细胞系的克隆形成，这是测试电离辐射效应的先决条件。不幸的是，细胞系不能形成足够的集落。第三个细胞系(UCH-1的衍生品)被认为是形成集落的细胞系，但被发现不足以满足我们的目的。在这两种细胞系中，通过免疫印迹和免疫沉淀，我们证实了人类T的高水平表达。实验表明，抗T shRNA处理显著减缓了脊索瘤细胞系的增殖。适当的检测证实了T基因和蛋白的下调。基因芯片分析是比较抗T shRNA处理的细胞株和对照shRNA处理的细胞株目前正在进行中，预计在一个月内完成。此外，还对脊索瘤细胞的基础microRNA环境进行了分析。通过shRNA下调T的细胞系也将很快完成这一工作。最后，我们还鉴定了脊索瘤细胞系在电离辐射处理后表达发生变化的microRNA。类似实验的基因芯片分析也在进行中。

In the second year of this project, we have executed the Materials Transfer Agreements necessary for us to obtain the published chordoma cell lines, including a new cell line UCH-2. We have tested the two known chordoma cell lines for colony formation, which is a prerequisite for testing the effect of ionizing radiation. Unfortunately, the cell line does not form adequate colonies. A third (derivative of UCH-1) cell line promised as a line that forms colonies, was found not to be adequate for our purposes. In both cell lines, using western blotting and immunoprecipitation, we have confirmed high-level expression of human T. Experiments have shown that chordoma cell line proliferation is significantly slowed by anti-T shRNA treatment. Appropriate assays confirm the downregulation of T mRNA and protein. cDNA microarray analysis is to compare cell lines treated with anti-T shRNA with control shRNA treated cell lines are currently underway and is expected to be completed within a month. Furthermore, the baseline microRNA environment of chordoma cells has been analyzed. That for cell lines downregulated for T by shRNA will soon be completed as well. Finally, we have also identified microRNA's that are altered in their expression after ionizing irradiation treatment of chordoma cell lines. cDNA microarray analysis of similar experiments are under way.