过表达人细胞S期激酶相关蛋白2(Skp2)促进MCF-7乳腺癌细胞生长及S期细胞增加  

Over-expression of human S-phase kinase-associated protein 2( Skp2 ) promotes proliferation of MCF-7 breast cancer cells and increases the number of S phase cells

作　　者：杨成成[1] 南克俊[1] 秦思达[2] 

机构地区：[1]西安交通大学医学院第一附属医院肿瘤内科,陕西西安710061 [2]西安交通大学医学院第一附属医院胸外2科,陕西西安710061

出　　处：《细胞与分子免疫学杂志》2015年第10期1306-1310,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81402506)

摘　　要：目的构建带FLAG标签的细胞S期激酶相关蛋白2(Skp2)基因真核表达载体pc DNA3-FLAG-Skp2并检测其对MCF-7乳腺癌细胞生长及细胞周期的影响。方法采用反转录PCR从MDA-MB-231乳腺癌细胞中扩增Skp2基因,构建重组真核表达载体pc DNA3-FLAG-Skp2。酶切及测序鉴定重组质粒正确后,脂质体介导转染至MCF-7乳腺癌细胞中。采用Western blot法检测FLAG-Skp2的表达,Alamar blue比色法绘制细胞生长曲线,流式细胞术检测细胞周期。结果 PCR扩增片段与预期片段大小相符,测序结果与Gen Bank结果一致。Western blot结果显示质粒转染48 h后,细胞成功表达融合蛋白FLAG-Skp2。过表达Skp2的MCF-7乳腺癌细胞较对照组生长速度明显加快,S期细胞显著增多。结论成功构建人Skp2真核表达载体pc DNA3-FLAG-Skp2。过表达Skp2的MCF-7细胞生长加快,S期细胞增多。Objective To construct a eukaryotic expression vector for the human gene S-phase kinase-associated protein 2 （Skp2） with a FLAG tag （pcDNA3-FLAG-Skp2） and detect the effect of Skp2 over-expression on the cell growth and cell cycle in MCF-？ breast cancer cells, Methods Skp2 was amplified from MDA-MB-231 breast cancer cells by reverse-transcription polymerase chain reaction （RT-PCR） and then used to construct the eukaryotic expression vector pcDNA3-FLAG-Skp2. Integration of Skp.？. into the vector was confirmed via restriction digest and sequencing; The pcDNA3-FLAG-Skp2 was then transfected into MCF-？ breast cancer cells. Expression of Skp2 protein was verified by Western blotting. Cell growth was assessed by Alamar blue proliferation assay, cell cycle analysis was carried out by flow cytometry. Results The PCR amplified fragment was matched up with the anticipated result and its sequence was the same as the data published on GenBank, indicating that the recombinant plasmid pcDNA3-FLAG-Skp2 was constructed successfully. Western blotting revealed that the expression of Skp2 protein was markedly up-regulated in the pcDNA3-FLAG-Skp2 transfected MCF-7 cells at 48 hours. Furthermore, cell growth was significantly promoted in Skp2 over-expressed MCF-7 cells, and the cell count in S phase were also raised. Conclusion The recombinant eukaryotic expression vector pcDNA3-FLAG-Skp2 has been constructed and expressed in MCF7？ breast cancer cells successfully. Over-expression of Skp2 resulted in the increased cell growth and number of S phase cells in Skp2 transfected MCF-7 cells.

关 键 词：S期激酶相关蛋白2(Skp2) 真核表达载体 MCF-7细胞 细胞周期 

分 类 号：R730.54[医药卫生—肿瘤] R392-33[医药卫生—临床医学]