【作者】 赖国旗；

【导师】 邱宗荫；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2006， 博士

【摘要】 肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis-inducing ligand，TRAIL)是继TNF、FasL之后发现的肿瘤坏死因子超家族新成员，具有选择性地诱导癌化或转化的细胞发生凋亡，对正常细胞及人体组织安全无毒，同时，TRAIL可以不依赖p53。因此，TRAIL受到了国内外学者的广泛重视，有望成为新的抗肿瘤药物。 然而，目前，TRAIL对宫颈癌的作用机理并不十分清楚，仅停留于对某一种或某几种蛋白质的功能研究，难以在特定的时间和特定的空间整体地、系统地、透彻地阐释TRAIL的作用机制。本研究以HPV18阳性的宫颈癌细胞系HeLa为模型，通过原核表达系统表达的TRAIL蛋白，开展了TRAIL蛋白的生物学活性，作用靶位及作用机理的初步研究，总的结果是为TRAIL的作用靶点及作用机理提供新的理论依据。 我们的研究分四个部分进行。 第一部分 人可溶性TRAIL cDNA的克隆及原核表达系统的构建 利用基因工程技术克隆人可溶性TRAIL胞外区114～281片段的cDNA及构建其原核表达载体pET28c(+)-sTRAIL。将pET28c(+)-sTRAIL转化大肠杆菌BL21(DE3)，经氨苄西林筛选，建立原核表达系统pET28c(+)-sTRAIL/BL21(DE3)，该系统经用1.0mmol／LIPTG诱导4h，得获了TRAIL融合蛋白，纯化并鉴定，表明，其分子量约为19.8kD。 第二部分 TRAIL对HeLa生物活性的研究 用TRAIL蛋白干预HeLa，经过MTT法检测细胞存活分数；AO／EB更多还原

【Abstract】 Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the membrane-binding cytokines that belongs to the family of tumor necrosis factor (TNF). TRAIL has been shown to be a potent apoptosis inducer in a wide variety of cancer cells in vitro and to limit tumor growth efficiently in vivo without damaging normal tissues dependent on or not p53. These functions of TRAIL suggest that it might be a potential therapeutic agent to treat human cancers.However, up to date, the role of TRAIL in cervical cancer is not very clear except functional studies of certain proteins. It’s difficult to thoroughly explain the role of TRAIL, in a particular time and specific space. In this paper, first , we have cloned cDNA of human TRAIL, and obtained TRAIL protein in the prokaryotic expression system . Next, with HPV18-positive cervical cancer cell lines HeLa as model, we have studied the biological activity of TRAIL, target proteins by Comparative Proteomics. The overall results laid a solid theoretical foundation for further research on the role and mechanisms of TRAIL .Our study is composed of four contents.Part I Cloning of human soluable TRAIL and expression in prokaryotic expression system .By means of genetic engineering technology, TRAIL codons 114～118 gene were amplified by RT-PCR, transformed into coli BL21 (DE3), and更多还原