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Analysis of gene alteration in volved in endometrial carcinogensis using in vitro model of human normal endometrial glandular cells

利用人正常子宫内膜腺细胞体外模型分析子宫内膜致癌相关基因改变

基本信息

批准号：
14370527
负责人：
SHIOZAWA Tanri
金额：
$ 8.9万
依托单位：
Shinshu University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370527/
关键词：
endometrium endometrial carcinoma immortalization large T antigen telomerase subtraction microarray Ras エストロゲン

项目摘要

The molecular mechanisms of endometrial carcinogenesis have not fully been understood. To analyze the pathogenesis of endometrial carcinoma in vitro, we planned to establish immortalized endometrial glandular cell lines. We first established an in vitro culture system of the normal endometrial glandular cells and we then transfected a large T antigen expression plasmid to the cultured cells. The transfected cells successfully survived until 20th passage of culture. We are now in preparation to transfect the large T antigen using viral vectors for better transfection efficiency to obtain longer viable passages.To discover new gene alterations involved in endometrial carcinogenesis, we isolated mRNA from frozen tissue sections of the normal, hyperplastic and malignant endometrial glands of the identical patients using laser-captured microdissection. The isolated mRNA was then amplified using T7-RNA polymerase-based amplification, and the amplified mRNA were subjected to cDNA subtraction and microarray to detect genes whose expression differ among the normal, hyperplastic and malignant tissues. Subsequently, we cloned approximately 30 new genes which are overexpressed in neoplastic tissues compared to the normal counterparts, and approximately 20 new genes which are down-regulated in the neoplastic lesions. The tissue specificity of most of these gene were confirmed by semi-quantitative RT-PCR. We are currently investigating the further tissue specificity of the expression of these genes using in situ hybridization.

子宫内膜癌发生的分子机制尚未完全了解。为探讨子宫内膜癌的发病机制，本研究拟建立永生化子宫内膜腺上皮细胞系。我们首先建立了正常子宫内膜腺细胞的体外培养体系，然后将大T抗原表达质粒转染到培养的细胞中。转染细胞成功存活至第20代。我们现在正在准备使用病毒载体进行大T抗原的转染，以获得更好的转染效率，以获得更长的可行passenges.To发现新的基因改变参与子宫内膜癌的发生，我们分离mRNA从冷冻组织切片的正常，增生和恶性子宫内膜腺体的相同患者使用激光捕获显微切割。采用T7-RNA聚合酶扩增技术对mRNA进行扩增，并将扩增的mRNA进行cDNA差减和基因芯片分析，以检测正常组织、增生组织和恶性组织中表达差异的基因。随后，我们克隆了约30个新的基因，这些基因在肿瘤组织中过表达，与正常对照相比，约20个新的基因在肿瘤病变中下调。半定量RT-PCR结果表明，大部分基因具有组织特异性。我们目前正在研究进一步的组织特异性，这些基因的表达，使用原位杂交。