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

子宫内膜癌发生的分子机制尚未完全了解。为了在体外分析子宫内膜癌的发病机制，我们计划建立永生化子宫内膜腺细胞系。我们首先建立了正常子宫内膜腺细胞的体外培养系统，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培养第20代。我们现在正准备用病毒载体转染大T抗原，以提高转染效率，获得更长的存活传代。为了发现参与子宫内膜癌变的新基因改变，我们使用激光捕获显微解剖技术从同一患者的正常、增生和恶性子宫内膜腺的冷冻组织切片中分离mRNA。然后用T7-RNA聚合酶扩增分离的mRNA，对扩增的mRNA进行cDNA减法和芯片检测正常组织、增生组织和恶性组织中表达差异的基因。随后，我们克隆了大约30个在肿瘤组织中比正常组织过表达的新基因，以及大约20个在肿瘤病变中下调的新基因。其中大部分基因的组织特异性经半定量RT-PCR证实。我们目前正在使用原位杂交技术进一步研究这些基因表达的组织特异性。

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.