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Role of cyclin E as an oncogene for human gastrointestinal tract cancer

细胞周期蛋白 E 作为人类胃肠道癌癌基因的作用

基本信息

批准号：
09670186
负责人：
YOKOZAKI Hiroshi
金额：
$ 1.73万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

1. Construction of cyclin E expression vector and introduction to mouse NIH/3T3 fibroblast(1) A 2.5 Kbp fragment of cyclin E cDNA that contained its total open reading frame was subcloned in to EcoRI site of mammalian expression vector pCDNA3 with cytomegalovirus promotor.(2) The cyclin E expression vector or mock pCDN3 were transfected to mouse NTH/3T3 fibroblast by lipofection method. After G418 selection, each 10 clones were recovered. Integration of the vectors and expression of recombinant cyclin E protein were confirmed by Southern and Western blot analysis, respectively.(3) Significant difference in growth rates were not observed between cyclin E transvectant and mock transfectant. No transformation focus was detected in cyclin E transfectants.2. Construction of cyclin E antisense vector and introduction to human gastric cancer cell lines.(1) A 11theta bp cyclin E cONA fragment was subcloned in antisense direction to pCDNA3 vector.(2) antisense cyclin E vector was transfected into human gastric cancer cell lines (TMK.-1, MKN-7, MKN-28 and MKN-74). Each 10 G418 resistant clones were recovered.(3) Significant difference in growth rates were not observed between antisense cyclin E transfectant and mock transfectant.These in vitro observations in the present research project provided any direct evidence supporting the idea that cyclin E can act as oncogene in human gastrointestinal carcinogenesis.

1. 细胞周期蛋白E表达载体的构建及小鼠NIH/3T3成纤维细胞的导入(1)将含有总开放阅读框的细胞周期蛋白E cDNA 2.5 Kbp片段亚克隆到含有巨细胞病毒启动子的哺乳动物表达载体pCDNA3的EcoRI位点。(2)采用脂肪转染法将cyclin E表达载体或模拟pCDN3转染小鼠NTH/3T3成纤维细胞。选择G418后，各恢复10个无性系。通过Southern和Western blot分析证实了载体的整合和重组cyclin E蛋白的表达。(3) cyclin E转染物与模拟转染物的生长速率无显著差异。cyclin E转染体未检测到转化灶。细胞周期蛋白E反义载体的构建及其在人胃癌细胞系中的导入。(1)将11theta bp cyclin E cONA片段反义克隆到pCDNA3载体上。(2)将反义细胞周期蛋白E载体转染人胃癌细胞系（TMK）。-1, MKN-7， MKN-28和MKN-74)。各获得10个G418抗性无性系。(3)反义cyclin E转染株与模拟转染株的生长速率无显著差异。本研究项目的这些体外观察提供了任何直接证据来支持细胞周期蛋白E在人类胃肠道癌变中作为致癌基因的观点。