腺病毒介导转铁蛋白受体报告基因在结直肠癌细胞MRI中的初步观察

Preliminary investigation on adenovirus-mediated transferrin receptor reporter gene in colorectal cancer cell MR imaging

目的探讨腺病毒载体介导转铁蛋白受体报告基因(TFRC)转染人结直肠癌Lovo细胞的最佳感染复数(MOI)、目标基因表达情况及体外MR成像情况。方法采用介导转铁蛋白受体报告基因的腺病毒(Ad-TFRC)载体以5、10、50、100的MOI转染Lovo细胞,以实时定量PCR检测相应mRNA表达。以浓度不一的转铁蛋白(Tf)-超微超顺磁性氧化铁(USPIO)探针转染细胞后,经普鲁士蓝染色确定最佳浓度,并以台盼蓝染色评价细胞活力。采用7.0T MR以T2、T2map及T2~*map序列对目标细胞成像,分析信号强度差异。结果 Ad-TFRC成功转染Lovo细胞,最佳MOI值为50,感染效率>90%;实时定量PCR检测显示Ad-TFRC转染Lovo细胞中TFRC蛋白表达量较空白对照Lovo细胞增多(P<0.01);普鲁士蓝染色结果示探针铁浓度为1.5μg/ml时最佳,Lovo细胞内可见大量蓝染铁颗粒;探针标记的Lovo细胞及空白对照Lovo细胞盼蓝染色拒染率分别为(93.80±1.60)%和(95.10±2.30)%,差异无统计学意义(P>0.05);体外MR T2W、T2map及T2~*map成像显示,与空白对照Lovo细胞比较,探针标记的Lovo细胞信号强度减弱(P<0.05)。结论腺病毒载体可在Lovo细胞中高效表达转铁蛋白受体报告基因,磁化标记后可成功实现Lovo细胞的体外7.0T MR显像。

Objective To explore the best multiplicities of infection（MOI）,the expression of the target gene and in vitro MR imaging of adenovirus vector-mediated transferrin receptor（TFRC）reporter gene transfection of human colorectal cancer Lovo cells.Methods Lovo cells were transfected with recombinant adenovirus（Ad-TFRC）at 5,10,50,100 MOI to determine the best MOI,and quantitative real-time PCR was performed to detect the cDNA of TFRC.The transfected cells were incubated in the culture medium including Tf-USPIO of various concentrations,and were observed by Prussian blue staining,then the cell viability was evaluated via Trypan blue staining.The labeled cells were scanned with 7.0 T MR T2 W,T2 map,T2~＊map sequences,and the signal intensities were analyzed.Results Ad-TFRC were successfully transfected into Lovo cells.The best MOI was 50,and the efficiency of infection was more than 90%.The relative expression amount of TFRC in transfected cells was higher than that in control Lovo cells by real-time quantitive PCR（P〈0.01）.Prussian blue staining showed numerous blue iron particles in transfected cells when the best labeling concentration was 1.5μg/ml.Trypan blue staining results of transfected Lovo cells and control Lovo cells was（93.80±1.60）% and（95.10±2.30）%,respectively（P〈0.05）.MR imaging in vitro showed that compared with control Lovo cells,the signal intensity decreased on T2 WI,T2 map and T2~＊map sequences in transfected Lovo cells（P〈0.05）.Conclusion TFRC reporter gene can be efficiently mediated by adenovirus for expression in Lovo cells.After magnetization labeling,7.0 T MR imaging of Lovo cells can be successfully achieved in vitro.