重组腺病毒介导的转录因子Runx3在神经胶质瘤细胞中的表达及其亚细胞定位  

Expression and localization of adenovirus-mediated transcriptional factor Runx3 in malignant glioblastoma cells

作　　者：白文栋[1,2] 张瑞[1,3] 孟艳玲[1,2] 曹云新[1,2] 王涛[1,2] 赵晶[1,3] 杨安钢[1,2,3] 

机构地区：[1]第四军医大学国家肿瘤生物重点实验室 [2]第四军医大学免疫学教研室,陕西西安710032 [3]第四军医大学生物化学与分子生物学教研室,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2009年第10期866-869,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30873006);第四军医大学"青年英才支持计划"(2009)

摘　　要：目的:制备含有融合Flag标签的Runx3基因的复制缺陷型重组腺病毒,感染神经胶质细胞瘤U251,观察外源Runx3在细胞中的表达及亚细胞定位。方法:用PCR的方法扩增Runx3基因,并将Flag标签蛋白的编码基因与RUNX3基因进行融合,构建腺病毒穿梭载体pShuttle-CMV-Runx3,经KpnI/XhoI双酶切鉴定并测序。利用电转化方法将经PmeI线性化的pShuttle-CMV-Runx3穿梭载体导入BJ5183重组细菌,获取重组腺病毒质粒Ad-Runx3,再将经PacI线性化的Ad-Runx3重组病毒骨架质粒转染293A包装细胞,包装并扩增病毒。利用该病毒感染神经胶质细胞瘤U251,用免疫印迹法观察外源Runx3在细胞中的表达,用间接免疫荧光法观察其在细胞内的定位。结果:构建并包装表达Runx3蛋白的重组腺病毒,用重组腺病毒感染U251细胞后,经免疫印迹和间接免疫荧光法检测,可见外源导入的Runx3蛋白在细胞核内的特异性定位。结论:成功制备了含有融合Flag标签的转录因子Runx3基因的重组腺病毒,感染U251细胞,在细胞中观察到该分子表达后定位于细胞核中,为研究Runx3在神经胶质瘤发生中的作用奠定了实验基础。AIM： To construct the replicative defecient adenovirus Ad-Runx3 expressing Runx3, and to express it in U251 malignant glioblastoma cells. METHODS： The runx3 gene with a flag tag was amplified by PCR using pCMVS-AML2 as a template, and was confirmed by DNA sequencing. The adenovirus shuttle vector pShuttle-CMVRunx3 was constructed by introducing runx3 DNA fragment into the sites of Kpn Ⅰ and Xho Ⅰ of pShuttle-CMV vector. This recombinant plasmid was linearized by Pmel and electronically transfected into BJ5183 cells to get the recombinant adenovirus vector Ad-Runx3. The recombinant adenovirus expressing Runx3 was infected into U251 malignant glioblastoma cells. The expression of exogenous Runx3 was observed by immonoblotting and its localization was detected by immunostaining using anti-Flag tag antibody. RESULTS： The recombinant adenovirus expressing Runx3 with a Flag tag was constructed and infected into U251 glioblastoma cells. The exogenous Runx3 protein was detected only in the nuclei. CONCLUSION： The recombinant adenovirus expressing Runx3 with a Flag tag is constructed successfully, and the Runx3 protein expressed in the nuclei of infected cells. The study laid a foundation for further research of the function of Runx3 in gliocarcinogenesis.

关 键 词：RUNX3 腺病毒 恶性脑胶质瘤细胞 

分 类 号：R392.11[医药卫生—免疫学]