人丝氨酸-苏氨酸激酶1过表达慢病毒细胞株构建以及功能检测  

Construction of human serine/threonine kinase 1 overexpressed cell line and function detection of serine/threonine kinase 1

作　　者：徐卿 李洪涛[2] 林峰[2] 勘b阳 

机构地区：[1]苏州大学医学部,215006 [2]上海交通大学附属第六人民医院肿瘤科,200030

出　　处：《中华实验外科杂志》2018年第2期323-325,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金青年科学基金（81102038）

摘　　要：目的构建带绿色荧光蛋白（GFP）标签的丝氨酸-苏氨酸激酶1（LKB1）慢病毒过表达细胞株，观察其在哺乳动物细胞内的表达和定位以及相关功能影响。方法构建出慢病毒表达质粒pCDH-CMV-MCS-EF1-copGFP载体（pCDH-LKB1）并筛选慢病毒细胞株。运用免疫荧光及Western blot检测其在哺乳动物细胞中的定位与表达。同时使用细胞计数试剂盒（CCK-8）检测LKB1对细胞活性的影响。结果成功构建LKB1慢病毒细胞株，荧光定位实验表明LKB1在人成骨肉瘤细胞细胞的细胞质细胞核均有分布；Western blot法检测表明LKB1在真核细胞中能有效表达。CCK-8检测显示，转染48h后，对照组1.131±0．003，LKB1组0．739±0．051，LKB1组相比于对照组，活性降低，两组差异有统计学意义（P=0．001）。结论成功构建过表达带绿色荧光蛋白标签的LKB1蛋白（GFP-LKB1）的细胞株，LKB1过表达明显抑制肿瘤细胞的活性。Objective -The purpose of this study is construct the lentivirus vector and over expressing cell line of human serine/threonine kinase 1 （ LKB1 ） with green fluorescent protein （GFP） tag, and study the normal expression and location of LKB1 protein on osteosarcoma cell U2OS cell line. Methods Human cDNA containing the full - length of LKB1 was used as template for PCR to construct GFP - LKB1 lentivirus vector. The location and expression of LKB1 in mammalian cells were detected by immunoflnorescence and Western blotting assay. Results The lentivirus plasmid and over expressing cell line of LKB1 was constructed successfully, and fluorescence localization test data analysis showed that the human LKB1 protein localizes both in cytoplasm and nucleus. Western blotting test show that LKB1 protein can over expression in Eukaryotic cells. CCK- 8 detection showed that after 48 hours of transfection, when control group （1. 131 ±0. 003 ） versus LKB1 group （0. 739 ± 0. 051 ） that LKB1 group＇s activity decreased, and the two groups had a distinct statistical significance. Conclusion In this study, we successfully constructed a cell line overexpressing GFP- LKB1 and LKB1 overexpression significantly inhibited the activity of tumor ceils, which provide a foundation for further study on the function and interaction partners of LKB1 protein.

关 键 词：骨肉瘤 丝氨酸-苏氨酸激酶1 

分 类 号：R738.1[医药卫生—肿瘤]