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Isolation and characterization of new markers for human brain tumors using molecular biological techniques

使用分子生物学技术分离和表征人脑肿瘤新标记物

基本信息

批准号：
06557078
负责人：
USUI Hiroshi
金额：
$ 11.33万
依托单位：
Niigata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06557078/
关键词：
brain tumor tumor marker gene expression cDNA cloning brain development onco-fetal antigen glioma genemutation onco‐fetal antigen differential screening

项目摘要

In this research project, we first established improved molecular biological procedures for efficient isolation of cDNA clones of differentially expressed mRNAs from small materials : (1) modified differential screening procedure, (2) directional tag PCR subtraction, an efficient subtractive cDNA cloning procedure, and (3) cDNA library DNA-Southern blot procedure which can replace Northern blot analysis.Using these procedures, we successfully isolated 22 distinct rat cDNA clones whose corresponding mRNAs were preferentially expressed in the fetal brain during the brain development. We then isolated their human homologues and analyzed the mRNA expressions in various human tissues. We found that the expression pattern of the human neuronatin was notable. The neuronatin mRNA was selectively expressed in fetal human brain among normal human tissues and in human pituitary adenomas among various human brain tumors, suggesting the utilization as a new marker for the pituitary adenomas. We also analyzed the mRNA patterns of human glioma cell lines exhibiting different biological characters using the procedures described above. We identified 9 cDNA clones whose corresponding mRNAs were expressed more abundantly in the cells which showed hetero-transplantability and higher growth rate than the control cells. These cDNA clones included novel clones and those involved in the protein synthesis, signal transduction, and nucleotide metabolism. These findings showed that our improved procedures are useful to analyze molecular basis of tumor cells and to identify new tumor markers.In addition, we analyzed gene mutations in human brain tumors to understand the molecular backgrounds of different mRNA expressions, and found frequent gene mutations of tumor suppressor genes, such as p53 and p16, in human brain tumors.

在本研究项目中，我们首先建立了改进的分子生物学程序，用于从小材料中有效分离差异表达mRNA的cDNA克隆：（1）改良的差异筛选法，（2）定向标签PCR消减法，一种有效的消减cDNA克隆法，（3）cDNA文库DNA-Southern印迹法，它可以取代北方印迹分析。我们成功地分离了22个不同的大鼠cDNA克隆，其相应的mRNA在脑发育过程中优先在胎儿脑中表达。然后，我们分离了它们的人类同源物，并分析了它们在各种人体组织中的mRNA表达。我们发现人神经元素的表达模式是显著的。neuronatin mRNA在正常组织中选择性地表达于胎儿脑，在多种脑肿瘤中选择性地表达于垂体腺瘤，提示neuronatin mRNA可作为垂体腺瘤的一个新的标志物。我们还使用上述程序分析了表现出不同生物学特性的人胶质瘤细胞系的mRNA模式。我们鉴定了9个cDNA克隆，其相应的mRNA在显示异源移植性和比对照细胞更高的生长速率的细胞中更丰富地表达。这些cDNA克隆包括新的克隆和那些参与蛋白质合成，信号转导和核苷酸代谢。此外，我们还对人脑肿瘤中的基因突变进行了分析，以了解不同mRNA表达的分子背景，并发现了人脑肿瘤中常见的抑癌基因p53和p16的基因突变。