摘要
目的细胞角蛋白7(Cytokeratin-7,Krt7)与肿瘤发展以及炎症反应密切相关,氧化应激在此过程中扮演着重要的角色,文中旨在通过构建小鼠Krt7的真核表达载体,并观察其在NIH 3T3细胞中的表达、定位以及在氧化应激反应下的变化情况。方法提取C57/BL6小鼠肺组织的总RNA,通过RT-PCR扩增得到Krt7编码序列,构建重组质粒pcDNA3-Krt7-HA。经脂质体转染NIH 3T3细胞,采用Western blot检测Krt7表达,免疫荧光检测其在细胞中的表达、定位以及在过氧化氢(H2O2)刺激下的变化情况。结果重组质粒构建成功。Western blot显示该质粒能够在NIH 3T3细胞中有效表达,免疫荧光检测表达产物主要分布在细胞质中,随着H2O2刺激时间的增加细胞内颗粒状蛋白聚集物的形成愈发明显且在60 min处发生入核现象。MEK通路抑制剂PD98059可明显抑制这一过程。结论成功构建了带HA标签的Krt7真核表达载体,该载体能在哺乳动物细胞中有效表达并正确定位,H2O2诱导Krt7在NIH 3T3细胞内的形态及定位变化依赖于MEK介导的信号通路。
Objective Cytokeratin-7 （Krt7） is closely related to tumor progression and inflammatory response, and in this process, oxidative stress plays an important role. By constructing a eukaryotic expression vector of Krt7, the authors observed the expres- sion and localization of Krt7 and its changes under oxidative stress in NIH 333 cells. Methods Total RNA was extracted from the lung tissue of C57/BI,6 mice, and the corresponding coding sequences of mouse Krt7 were amplified by RT-PCR and then cloned into the he- magglutinin （HA）-tagged vector pcDNA3-HA to construct a new recombinant plasmid named pcDNA3-Krt7-HA. The NIH 313 cells were then transfected with liposome. The expression of Krt7 was determined by Western blot. The distribution and changes of the expressed products induced by hydrogen peroxide in the transfected NIH 3T3 cells were observed by immunofluorescence. Results The recombi- nant plasmid was correctly constructed, and it was found to be effectively expressed in the NIH 3T3 cells and distributed mainly in the cy- toplasm. With the increased time of H2 02 stimulation, intracellular granular protein aggregates became more apparent and were transloca- ted from cytoplasm to the nucleus at 60 rain. And this process was obviously inhibited by the inhibitor of the MEK signaling pathway PD98059. Conclusion The eukaryotic expression vector of the KrtT-HA fusion protein was successfully constructed and effectively ex- pressed in mammalian ceils with a correct localization. The morphology and localization of Krt7 in NIH 333 cells induced by H202depend on the MEK signaling pathway.
出处
《医学研究生学报》
CAS
北大核心
2013年第8期789-793,共5页
Journal of Medical Postgraduates
基金
国家自然科学基金(81030055)
广东省自然科学基金(10251051501000003)
教育部长江学者和创新团队发展计划项目(IRT0731)
关键词
细胞角蛋白
分子克隆
氧化应激
信号通路
Cytokeratin
Molecular cloning
Oxidative stress
Signaling pathway
