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The basic pathological research for the establishment of the diagnoses of bone tumors and soft tissue tumors using DNA microarray.

利用DNA微阵列建立骨肿瘤和软组织肿瘤诊断的基础病理学研究。

基本信息

批准号：
13670164
负责人：
MATSUBAYASHI Jun
金额：
$ 2.3万
依托单位：
Tokyo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670164/
关键词：
Soft tissue tumor Bone tumor Microarray Chimeric gene Chromosomal translocation RT-PCR Genetic diagnosis

项目摘要

In this study, we reevaluated about 100 cases of bone tumors and soft tissue tumors whether they had been histologically diagnosed correctly or not. We performed chromosomal analysis and molecular analysis of chromosomal translocations about some cases of diem. For example, a chimeric gene : EWS-ATF1 was identified in all of 8 cases of clear cell sarcoma by RT-PCR. On the basis of the results, we performed the basic experiment for construction of microarray to detect chimeric genes of bone tumors and soft tissue tumors. At first, we examined synovial sarcoma (SS) cases, that were divided into only two subtypes and of which breaking points were simple. We would have independently detected SYT-SSX1, SYT-SSX2 in each SS cases by multiplex RT-PCR method. Moreover, we made a microarray that had three genes: SYT, SSX1, SSX2 and hybridized cDNA fragments amplified by PCR. On the array, the cDNA fragments reacted with the gene arrangement as same as the specific chimeric gene, but didn't reacted with the gene arrangements as same as other chimeric genes. Additionally, we examined EWS-related chimeric genes about EWS-related tunors by both conventional RT-PCR method and multiplex RT-PCR method. As a result, the results by conventional RT-PCR method were as same as the results by multiplex RT-PGR method in many cases. But we couldn't identify target chimeric genes in some cases. And we also had non-specific reactions in some cases. We speculated that these results were due to number of cycle of PCR and reaction temperature. Thus we found, that it is difficult to set up the best condition of the multiplex RT-PCR. But we also found that these results were important and basic data to reach the foundation of the genetic diagnosis of the bone tumors and soft tissue tumors that had chimeric genes, using mnicroarray method.

在这项研究中，我们重新评估了约100例骨肿瘤和软组织肿瘤，无论它们是否被组织学诊断正确。我们对部分diem病例进行了染色体分析和染色体易位的分子生物学分析。例如，通过RT-PCR在8例透明细胞肉瘤中均检测到嵌合基因EWS-ATF 1。在此基础上，我们进行了构建骨肿瘤和软组织肿瘤嵌合基因芯片的基础实验。首先，我们研究了滑膜肉瘤（SS）的情况下，分为只有两个亚型，其中断点是简单的。采用多重RT-PCR方法分别检测每例SS患者的SYT-SSX 1、SYT-SSX 2。此外，我们制作了一个芯片，其中有三个基因：SYT，SSX 1，SSX 2和杂交的cDNA片段通过PCR扩增。在芯片上，cDNA片段与特定嵌合基因的基因排列反应，而与其他嵌合基因的基因排列不反应。此外，我们还用常规RT-PCR和多重RT-PCR方法检测了EWS相关嵌合基因。结果表明，常规RT-PCR方法与多重RT-PCR方法在多数情况下检测结果一致。但在某些情况下，我们无法识别目标嵌合基因。在某些情况下，我们也有非特异性反应。我们推测这些结果是由于PCR循环次数和反应温度。因此，我们发现，建立多重RT-PCR的最佳条件是困难的。但我们也发现，这些结果是重要的和基础的数据，达到的基因诊断的骨肿瘤和软组织肿瘤的嵌合基因，利用微阵列方法。