Isolation of genes related to malignant transformation of thyroid tumors and gene therapy for undifferentiated carcinoma of the thyroid

甲状腺肿瘤恶变相关基因的分离及甲状腺未分化癌的基因治疗

• 批准号: 07671143
• 负责人: YOSHIMOTO Katsuhiko
• 金额: $ 1.6万
• 依托单位: The University of Tokushima
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07671143/
• 关键词: thyroid tumors; malignant transformation; mRNA; differential display; AP-PCR; 欠喪; 増幅; differential display法

To elucidate the molecular basis for malignant transformation of thyroid tumors, changes in mRNA and genome were analyzed with the differential display method and arbitrarily primed (AP)-PCR method.1. Differential display methodRNA was extracted from a thyroid follicular adenoma cell line, three thyroid papillary carcinoma cell lines, a thyroid follicular carcinoma cell line, and two undifferentiated thyroid carcinoma cell lines. Bands with different strength of signals among cell lines in the differential display method were marked in the autoradiography and cut out from the gels. They were amplified, cloned, and sequenced. Half of the clones were the known genes such as ribosome RNA,mitochondrial gene, fibronectin, R-ras, a subunit of proteasome, 23 kD highly basic protein. Half of the clones were unknown genes. Northern blot analysis using these clones as probes showed that any clones have the expression levels less than 2-fold among cell lines.2. AP-PCR methodGenomic DNAs were extracted from 12 thyroid tumor cell lines. DNAs were amplified in conditions that an arbitrarily oligonucleotide with 20 uncleotides was used as a primer and DNA templates were amplified with the low annealing temperature in the first 5 cycles. Patterns of bands among cell lines in the autoradiograph were compared. A loss of signal suggesting deletion was found in a thyroid papillary carcinoma cell line, however, an apparent deletion was not detected in southern blot analysis using the cloned DNA as a probe. In addition, no signals suggesting gene amplification in cell lines were not found.

为了阐明甲状腺肿瘤恶性转化的分子基础，采用差异显示法和任意引物(AP)-PCR法分析了mRNA和基因组的变化。分化显示方法从1株甲状腺滤泡腺瘤细胞株、3株甲状腺乳头状癌细胞株、1株甲状腺滤泡癌细胞株和2株未分化甲状腺癌细胞株中提取rna。在放射自显影术中标记出细胞间信号强度不同的条带，并将其从凝胶中剪切出来。它们被扩增、克隆并测序。一半的克隆是已知的基因，如核糖体RNA、线粒体基因、纤维连接蛋白、R-ras（蛋白酶体亚基）、23kd高碱性蛋白。一半的克隆是未知的基因。用这些克隆作为探针进行Northern blot分析，结果表明，这些克隆在细胞系间的表达量均小于2倍。AP-PCR方法从12株甲状腺肿瘤细胞系中提取基因组dna。在前5个循环中，以任意寡核苷酸为引物，在低退火温度下扩增DNA模板。比较了射线自显影中细胞系间条带的形态。在甲状腺乳头状癌细胞系中发现了缺失信号的缺失，然而，使用克隆DNA作为探针，在southern blot分析中未检测到明显的缺失。此外，没有发现细胞系中基因扩增的信号。

项目成果

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