食管癌细胞中fascin基因启动子区的克隆与初步鉴定_医学论文.docVIP

食管癌细胞中fascin基因启动子区的克隆与初步鉴定_医学论文 食管癌细胞中fascin基因启动子区的克隆与初步鉴定_医学论文 作者：杨章民，郭张燕，许丽艳，高书颖，谢剑君，李恩民 【摘要】 　　目的：对食管癌细胞中fascin基因启动子区进行克隆鉴定. 方法：应用PCR技术从食管癌细胞系EC109中扩增出fascin基因5′侧翼区5段不同长度的片段，并将其克隆至萤火虫荧光素酶报告基因表达载体pGL3（pGLB）中, 然后将这些质粒与对照质粒pGLB、内参照质粒pRL共转染EC109细胞，48 h后用双荧光素酶检测系统分析这些重组子报告基因转染细胞的相对荧光强度，并结合生物信息学分析，以判断fascin基因启动子区所在位置. 结果：用DNA重组技术成功构建了5个系列缺失重组荧光素酶报告基因表达载体. 相对荧光素酶活性检测表明，与对照组相比，上述一系列重组质粒转染细胞均显示出较高的荧光素酶活性. 生物信息学分析发现，在启动子区(-436～+1)有标志性调控元件TATA盒(-34～-29). 结论：在食管癌细胞中fascin基因的启动子区可能位于转录起始位点上游约436 bp的区段内. 【关键词】 fascin基因； 食管肿瘤； 启动子； 荧光素酶类； 报告基因 　　【Abstract】 AIM: To clone and identify the promoter region of fascin gene in esophageal carcinoma cells. METHODS: Five difr cell line) by using PCR and cloned into luciferase reporter gene vector pGL3Basic （pGLB）, which aids in verification of functional promoter elements. Then the relative luciferase activities of the report genes in the EC109 cells transfected with these recombinant plasmids were analyzed by Dual Luciferase Reporter Gene Assay System (DLR). Bioinformatic analysis was also performed to determine the position of fascin gene promoter. RESULTS: Five serial deletion recombinant luciferase report gene expression vectors pBF-2900- -436 were successfully constructed by using DNA recombination technique. 48 h after cotransfection of these recombinant plasmids with control pGLB, pRLTK vector into EC109 cells, the relative luciferase activities of the cells transfected with these recombinant plasmids were all higher, as compared with controls. Bioinformatic analysis unveiled a TATA box (-34- -29), which was the marked regulatory element, in this promoter region (-436- +1). CONCLUSION: The promoter region of fascin gene is likely located in the region of 436 bp upstream of the transcriptional initiation site in esophageal carcinoma cells. 　　【Keywords】 fascin esophageal neoplasms promoter Luciferase Reporter Gene Assay 　　0 引言 　　研究表明，癌细胞侵袭转移的恶性行为是由于肌动蛋白结合蛋白等因素引起的细胞骨架异常重组所致［1］. 成束蛋白（fascin）是肌动蛋白结合蛋白中与癌细胞运动时丝状伪足、微棘异常形成有关的主要分