KPNA2表达载体的构建及其对骨肉瘤细胞生长的影响  

Construction of KPNA2 expression vector and its effects on the growth of osteosarcoma cells

作　　者：董文刚 刘锐[1] 王飞[1] 沈伟伟 黄强[1] 李闯斌 李文波[1] 王伟[1] 薛云 高秋明 Dong Wengang;Liu Rui;Wang Fei;Shen Weiwei;Huang Qiang;Li Chuangbin;Li Wenbo;Wang Wei;Xue Yun;Gao Qiuming(Department of Orthopedics, the 940 Hospital of Joint Logistics Support Force of PLA, Gansu Lanzhou 730050, China)

机构地区：[1]中国人民解放军联勤保障部队第940医院创伤骨科

出　　处：《现代肿瘤医学》2019年第17期2988-2991,共4页Journal of Modern Oncology

基　　金：国家自然科学基金青年基金(编号:81600700)

摘　　要：目的:通过分子克隆构建KPNA2过表达载体,探究其对骨肉瘤细胞143B生长的影响。方法:利用Trizol法提取Hela细胞总RNA,而后通过PCR反应扩增KPNA2基因,将扩增产物克隆至表达载体pcDNA3.1^+构建KPNA2过表达质粒。通过qPCR实验和Western blot实验检测KPNA2过表达载体转染143B细胞后的表达效率。利用CCK-8、平板克隆形成实验检测143B细胞的增殖状况,利用流式细胞术检测143B细胞的周期分布。结果:qPCR实验和Western blot实验均表明KPNA2过表达质粒转染后,143B细胞内KPNA2表达水平显著上调。CCK-8、平板克隆形成实验结果证实KPNA2上调能够促进143B细胞增殖。流式细胞术结果表明过表达KPNA2能够加快143B细胞周期进程。结论:KPNA2能够促进骨肉瘤细胞系143B生长,为骨肉瘤早期诊断和靶向治疗提供实验依据和理论基础。Objective:To construct KPNA2 expression vector with molecular cloning technology to upregulate the level of KPNA2 in osteosarcoma cell 143 B,and to investigate its effects on the growth of 143 B cells.Methods:Trizol was used to extract the total RNA of Hela cells and then RNA was reversely transcripted to cDNA.KPNA2 gene was amplified using PCR and cloned into the pcDNA3.1^+ vector to generate KPNA2 overexpression plasmid.The plasmid was transfected into 143 B cells.pcDNA3.1^+ vector group and blank group were regarded as negative control.Next,qPCR and Western blot were performed to detect the level of KPNA2 in 143 B cells.CCK-8 and colony formation assays were used to detected the cell proliferation of 143 B cells.FCM was performed to detect cell cycles of 143 B cells.Results:qPCR and Western blot results jointly showed transfection of KPNA2 overexpression plasmid resulted in upregulation of KPNA2 level in 143 B cells,compared with negative control.CCK-8 and colony formation assays results collectively indicated KPNA2 could promote the proliferation of 143 B cells.FCM results showed KPNA2 overexpression could accelerate cell cycles of 143 B cells.Conclusion:KPNA2 could obviously promote growth of 143 B cells,which may provide us with theoretical foundation for early diagnosis and targeted therapy of osteosarcoma.

关 键 词：KPNA2 骨肉瘤 表达载体 细胞生长 

分 类 号：R738.1[医药卫生—肿瘤]