稳定表达荧光素酶的nm23-H1表达缺失人肺腺癌细胞株的构建及其体内外活性检测  

Establishment of the Cell Line that Human Lung Adenocarcinoma can Stably Express Luciferase Which is Absent of nm23-H1 Expression and Detecting Its Luminescence in vitro and in vivo

作　　者：王红明[1,2] 朱大兴[1,2] 吴志浩[1,2] 周清华[1,2] 

机构地区：[1]天津市肺癌转移与肿瘤微环境重点实验室天津市肺癌研究所 [2]天津医科大学总医院,天津300052

出　　处：《中国肺癌杂志》2012年第3期152-158,共7页Chinese Journal of Lung Cancer

基　　金：国家自然科学基金重点项目(No.30430300);国家自然科学基金项目(No.30973384);国家"863"重大项目(No.2006AAOZA401);国家"973"重大项目(No.2010CB529405);中瑞国际合作项目(No.09ZCZDSF04100)资助~~

摘　　要：背景与目的在实验动物存活条件下,通过活体成像技术能探测到标记有萤火虫荧光素酶(luc)基因的肿瘤细胞在体内的分布情况。本研究旨在稳定表达nm23-H1 shRNA的人肺腺癌细胞株A549中,建立能稳定表达萤火虫荧光素酶的发光细胞株A549/nm23-H1-shRNA-luc,并检测其体内外发光情况,为下一步相关的体内实验提供实验材料。方法通过浓度梯度法测定A549/nm23-H1-shRNA细胞的潮霉素最佳筛选浓度,将带有萤火虫荧光素酶基因的PGL4.50质粒转入A549/nm23-H1-shRNA细胞中,利用潮霉素筛选单克隆细胞株A549/nm23-H1-shRNA-luc,并采用生物发光技术对单克隆细胞株进行阳性鉴定并挑选发光最强的1个克隆分析其表达荧光素酶的稳定性。为检测A549/nm23-H1-shRNA-luc细胞在体内发光的稳定性,将A549/nm23-H1-shRNA-luc细胞接种于10只裸鼠右后腹股沟皮下之后,并随机分为两组,每组5只裸鼠,运用活体成像系统观察成像情况。结果 A549/nm23-H1-shRNA-luc细胞的潮霉素最佳筛选浓度为300 g/mL。经过潮霉素筛选所建立的A549/nm23-H1-shRNA-luc细胞株能在体外稳定表达荧光素酶,细胞数(x)和生物发光值(y)呈直线相关,回归方程是:y=3,699.9x+992,237,R2=0.975,1。为评估此细胞株在体内生物发光的稳定性,将细胞种植进入10只裸鼠并随机分成两组,结果显示体内生物发光值差异不具有统计学意义(P>0.05)。结论成功建立了能持续、稳定表达荧光素酶的A549/nm23-H1-shRNA-luc细胞株。Background and objective On the condition that laboratory animals survive,we can detect the dis-tribution of tumor cells by in vivo imaging that were labeled with firefly-luciferase（luc）gene.The purpose of this study is to establish a light-emitting cell line A549/nm23-H1-shRNA-luc which could express nm23-H1 shRNA and firefly-luciferase sta-bly,and detect its bioluminescence in vitro and in vivo.It will provide the preparation for the next related experimental research in vivo.Methods The optimal concentration of hygromycin B for screening A549/nm23-H1-shRNA cells was determined by concentration gradient method.We firstly transfected the plasmid（PGL4.50）with luc gene into A549/nm23-H1-shRNA cells and then screened the monoclonal cell line A549/nm23-H1-shRNA-luc with hyhromycin B.The positive monoclonal cell line was identified with an in vivo imaging system,thereafter the expression stability of luciferase was analyzed in the strongest light-emitting positive monoclonal cell line.The A549/nm23-H1-shRNA-luc cells were inoculated subcutaneously into right-hind groin of nude mice and then observed by the in vivo imaging system.Results The optimal concentration of hygromycin B used in screening A549/nm23-H1-shRNA cells was 300 μg/mL.After screening,the A549/nm23-H1-shRNA-luc cells established can express luciferase stably in vitro,a great linear correlation existed between the amount of cells（x）and bioluminescence values（y）,with an equation of y=3,699.9x＋992,237,and the square of the correlation coefficient（R2）was 0.975,1.To evaluate the stability of bioluminescence in vivo,10 nude mice were randomly divided into two groups that the same number of cells were implanted into.The variation of bioluminescence values detected in vivo between the two groups of the same cells was not statistically significant（P0.05）.Conclusion We have successfully established the cell line A549/nm23-H1-shRNA-luc which can express luciferase persistently and stably.

关 键 词：荧光素酶 肺腺癌 生物发光成像 NM23-H1 

分 类 号：R734.2[医药卫生—肿瘤]