构建大肠癌细胞相关基因Nrf3融合蛋白真核表达载体及其体外转染的初步功能分析  

Constructing a fusion protein eukaryotic expression vector of colorectal cancer related gene Nrf3 and its initial functional study on transfection in vitro

作　　者：王强[1] 李晓利[1] 白雪娟[1] 黄仲曦[2] 崔春平[3] 丁彦青[2] 张金萍[4] 姚开泰[2] 许红民[5] 

机构地区：[1]解放军总医院第二附属医院器官移植中心,北京市100091 [2]南方医科大学病理解剖教研室及肿瘤研究所,广东省广州市510515 [3]解放军军事医学科学院放射与辐射医学研究所,北京市100850 [4]解放军第三○五医院心血管科,北京市100017 [5]解放军总医院医院病理科,北京市100853

出　　处：《中国组织工程研究与临床康复》2007年第37期7475-7480,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：全军"十五"医学科研基金重点项目(04Z041);北京市自然科学基金(7052064)~~

摘　　要：目的:应用基因芯片生物信息学分析思路筛查大肠癌细胞相关基因Nrf3,构建融合蛋白真核表达载体,检测其对体外转染癌细胞周期与凋亡的影响。方法:实验于2004-01/2006-07在南方医科大学病理解剖教研室及肿瘤研究所与中国农业大学农业生物技术国家重点实验室完成。①实验材料:大肠癌细胞组织及其配对的正常大肠黏膜各3例,由解放军总医院病理科提供;大肠癌LoVo细胞系由南方医科大学肿瘤研究所提供;肝癌SMMC7721细胞系由解放军军事医学科学院放射医学研究所提供;真核表达载体pEGFP-N1(Clontech公司)。②实验方法:分别应用Excel表、Affymetrix Microarray Suite Software 5.0分析软件和STATA7.0分析软件,对基因芯片表达谱数据行交集补集分析、秩和检验及T检验,筛选"最重要的大肠癌细胞差异表达基因&ESTs",差异表达P<0.05,差异倍数>2。应用文献轮廓法进一步分析确定首选研究基因为Nrf3,提取组织样品总RNA,cDNA合成,PCR扩增产物经琼脂糖凝胶电泳分离后回收纯化,插入T载体中,SalⅠ和BamHⅠ双酶切后连接到pEGFP-N1载体,获得重组pEGFP-N1-Nrf3质粒。LoVo细胞在体外加入含体积分数为0.1小牛血清、100u/mL青链霉素的RPMI-1640培养基,置于37℃、体积分数为0.05的CO2饱和湿度培养箱中,待转染质粒pEGFP-N1-Nrf3与阴性对照质粒pEGFP-N1转染36h后,荧光显微镜观察候选基因亚细胞定位,FCM分选术观察其在体外对LoVo细胞周期和凋亡的影响。同法观察重组pEGFP-N1-Nrf3质粒体外转染对SMMC7721细胞周期和凋亡的影响。结果:①首选研究大肠癌细胞相关基因的确认:多种统计学方法分析获得最重要的大肠癌细胞相关基因17个,文献轮廓法进一步确认Nrf3为首选研究基因。②Nrf3基因表达:RT-PCR法检测Nrf3在3例大肠癌细胞组织均有表达,而在与其相配对的正常黏膜中表达较弱或不表达,与芯片检测结果基本一致;Nrf3在LoVo细�AIM： Colorectal cancer （CRC） related gene Nrf3 was obtained using Genechip. The fusion protein eukaryotic expression vector was constructed. The cell cycle and apoptosis of cancer cells by the insertion of Nrf3 were checked in vitro. METHODS： The experiment had been completed in Cancer Institute ＆ Department of Pathology of Southern Medical University and National Laboratories for Agrobiotechnology of China Agricultural University from January 2004 to July 2006. ①Three CRC samples with 3 paired normal mucosa tissue were offered by Department of Pathology of General Hospital of Chinese PLA. The LoVo CRC cell line was provided by Cancer Institute ＆ Department of Pathology of Southern Medical University, SMMC7721 liver cancer cell line was offered by Institute of Radiation Medicine of Academy of Military Medical Sciences of Chinese PLA. The pEGFP-N1 was purchased from Clontech CO. ②Affymetrix oligonucleotide microarrays HG-U133 were analyzed to obtain candidate gene by kinds of statistics methods including T test, rank test, intersection and complement, and literature profiling using Excel, Affymetrix Microarray Suite Software 5.0 and STATA7.0 （differential expression P 〈 0.05, ratio 〉 2）. Nrf3 was identified by literal profiling. Total RNA was extracted from CRC and normal tissue to synthesize cDNA. The products of PCR were separated using agarose gel electrophoresis, then reclaimed and purification was completed. It was inserted into T trager and linked with pEGFP-N1 after diplo-enzyme using Sal Ⅰ and BamH Ⅰ in order to obtain pEGFP-N1-Nrf3 plasmid. The LoVo cell lines were cultivated in incubator of saturated humidity with 37 % and 0.05 volume fraction mark, after using RPMI-1640 medium with calf serum of 0.1 volume fraction mark and 100 u/mL mycillin in vitro. After 36 hours as pEGFP-N1-Nrf3 and pEGFP-N1 were transfected, the subcellular localization of Nrf3 gene were observed by Fluophot, at the same time, the call cycle and apoptosis of LoVo cell line were checked by flow cytometry

关 键 词：基因表达谱 Nrf3 质粒 大肠癌 流式细胞术 细胞周期 凋亡 

分 类 号：R735.34[医药卫生—肿瘤]