摘要
目的:建立一种基于分泌型萤光素酶的实时定量检测实验动物体内肿瘤大小的方法。方法:以分泌型Gaussia萤光素酶(Gluc)为报告基因,以嘌呤霉素为筛选基因,将两者用T2A元件连接后克隆到慢病毒载体,包装慢病毒后感染乳腺癌MCF-7细胞,经嘌呤霉素筛选得到稳定转染细胞MCF-7-Gluc,并检测细胞上清中Gluc活性随时间和细胞数目的变化;将MCF-7-Gluc扩大培养后经皮下注射到雌性BALB/c裸鼠前肢腋下,待肿瘤形成后,检测外周血液中Gluc活性与肿瘤体积的相关性。结果:体外实验显示稳定转染细胞MCF-7-Gluc分泌到细胞上清的Gluc活性与时间和细胞数量在一定范围内均呈现良好的线性关系,体内实验显示裸鼠血液中的Gluc活性与肿瘤体积呈正相关。结论:Gluc技术可作为一种灵活、方便、实时定量检测活体动物体内肿瘤大小的有效工具。
Objective： To establish a real-time quantitation method based on the secreting Gaussia luciferase （Gluc） that can detect the tumor size of live animals in vivo sensitively and conveniently. Methods： First, the secreting Gaussia luciferase reporter gene was fused to the puromycin gene by T2A element and cloned into the lentiviral vector. Lentivirus was packaged in 293T cells and infected the breast cancer cell MCF-7. The infected MCF-7 cells were screened by puromycin for several weeks and thus the stably transfected MCF-7-Gluc cells were obtained. The luciferase activity in the cell supernatant was detected over time and the number of ceils. The expanding cultured MCF-7-Gluc cells were implanted into the female nude mice by subcutaneous injections. Upon the formation of tumors, the luciferase activity correlated with the tumor volume was detected and analyzed. Resuits： In vitro experiments showed that the stably transfected MCF-7-GIuc cells had a good linear relationship between the cell supernatant luciferase activity and time/the number of ceils within a certain range; in vivo experiments showed that the serum luciferase activity of the BALB/c nude mice implanted with the stably transfected MCF-7-Gluc ceils was correlated positively with the tumor volumes. Conclusion： The secreting Gaussia luciferase can be used as a flexible and effective tool for real-time quantitation of hve animals＇ tumor size in vivo.
出处
《生物技术通讯》
CAS
2013年第3期370-373,共4页
Letters in Biotechnology
基金
国家自然科学基金(81202562)
国家重大新药创制专项(2012ZX09103301-044)
