Role of the human transcription factor T in chordoma growth

在该项目的第二年，我们签署了必要的材料转让协议，以获得已发表的脉络膜细胞系，包括新的细胞系UCH-2。我们已经测试了两种已知的脉络膜细胞系的集落形成，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。第三种（UCH-1的衍生物）细胞系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，使用蛋白质印迹和免疫沉淀，我们已经证实了高水平表达的人T。实验表明，抗T shRNA处理显著减缓了脉络膜细胞系的增殖。适当的测定证实了T mRNA和蛋白的下调。cDNA微阵列分析是比较用抗T shRNA处理的细胞系与对照shRNA处理的细胞系，目前正在进行中，预计将在一个月内完成。 此外，已经分析了脉络膜细胞的基线microRNA环境。通过shRNA下调T的细胞系也将很快完成。最后，我们还鉴定了在电离辐射处理脉络膜细胞系后其表达改变的microRNA。类似实验的cDNA微阵列分析正在进行中。

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.