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不完全清楚。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的子宫内膜腺细胞系。我们首先建立了正常子宫内膜腺细胞的体外培养体系，然后将一个大的T抗原表达载体导入培养的细胞。转染组细胞成功存活至第20代。我们现在正准备用病毒载体来转染大T抗原，以获得更高的转染效率，以获得更长的存活时间。为了发现与子宫内膜癌发生有关的新的基因变化，我们使用激光捕获显微切割技术从相同患者的正常、增生性和恶性子宫内膜腺的冰冻组织切片中提取了mRNA。以T7-RNA聚合酶为基础的扩增方法对分离的mRNA进行扩增，并对扩增出的mRNA进行cDNA消减和微阵列分析，以检测正常组织、增生性病变和恶性肿瘤组织中差异表达的基因。随后，我们克隆了大约30个与正常组织相比在肿瘤组织中过度表达的新基因，以及大约20个在肿瘤组织中下调表达的新基因。半定量RT-PCR证实了这些基因的组织特异性。我们目前正在用原位杂交的方法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.