电转系统介导的荧光素酶稳定表达PANC-1细胞的构建及其生物发光成像检测

Establishment of Pancreatic Cancer Cell Line Stably Expressing Luciferase Mediated by Nucleofector System and Detection with Bioluminescence Imaging System

在构建稳定表达荧光素酶的人胰腺癌PANC-1细胞的基础上,建立适合活体成像研究的胰腺癌动物模型,为深入研究胰腺癌发病机制打下基础。该研究运用Lonza NucleofectorTM核转染系统将携带有荧光素酶基因(firefly luciferase,luc)的质粒(GV258)稳定转染PANC-1细胞,利用嘌呤霉素筛选出能稳定表达荧光素酶的细胞株PANC-1-LUC,并应用活体成像系统对其体内外生物发光进行检测。进一步构建PANC-1-LUC裸鼠皮下移植瘤,同样方法接种慢病毒载体构建的PANC-1-luc细胞作为对照,观察成瘤率、瘤体积,检测生物发光强度。体内外生物发光检测结果均显示,光通量值与细胞数之间呈显著的直线相关(P<0.01)。PANC-1-LUC细胞皮下接种裸鼠,1周成瘤率即达80%以上,瘤体积可达平均100 mm3。观察6周,裸鼠皮下肿瘤生长符合Gompertzian曲线。每周的生物发光检测结果均显示,PANC-1-LUC移植瘤的光强度与瘤体积有显著的线性关系(P<0.05)。以上结果说明,该文所构建的PANC-1-LUC细胞株能够稳定、长期地表达荧光素酶,更适合构建活体成像研究的裸鼠胰腺癌模型。

The paper aimed to establish a pancreatic cancer cell line which could stably produce luciferase and suit for constructing xenotransplanted tumor model in nude mice. PANC-1 cells were transfected with plasmid （GV258） with luc gene mediated by Lonza NucleofectorTM System and monoclonal cell line PANC-1-LUC was screened with puromycin. The luciferase expression of PANC-1-LUC cells was detected by bioluminescence imaging system in vitro and in vivo. The results showed that the bioluminescence density of PANC-1-LUC cells correlated significantly to the cell numbers in vitro and in vivo （P〈0.01）. Furthermore, the PANC-1-LUC cells were inoculated subcutaneously into back of nude mice. A week later, the tumor growth and bioluminescence of xenotransplanted tumor were investigated. The high tumorigenecity above 80% was observed and the curve of tumor growth was accorded with Gompertzian curve. The results of weekly bioluminescence detection inferred a significant correlation between total flux value ofbioluminescence and volume of xenotransplanted tumor （P〈0.05）. Overall, we have successfully established a pancreatic cancer cell line, PANC-1-LUC, which could produce luciferase stably and persistently.