荧光素酶标记的EL4细胞的制备及淋巴瘤动物模型构建  

Preparation of Luciferase-labeled EL4 Cells and Model Establishment of Lymphoma-animal

作　　者：喻青[1] 

机构地区：[1]上海复旦大学附属中山医院青浦分院,上海201700

出　　处：《医学信息》2012年第8期84-86,共3页Journal of Medical Information

摘　　要：目的制备稳定表达萤火虫荧光素酶（Luciferase）小鼠T细胞白血病／淋巴瘤细胞系田L4），在细胞水平检测其生物发光能力；建立发光淋巴瘤动物模型，在整体动物水平观察肿瘤的生长及转移。方法通过阳离子脂质体法将含荧光素酶tuc）基因和绿色荧光蛋白（GFP）基因的慢病毒载体四质粒系统共转染入病毒包装细胞293T，24h后在荧光显微镜下观察GFP表达情况，监测转染效率。72h收集病毒上清，浓缩后以MOI=8感染EL4细胞，在荧光显微镜下和应用流式细胞仪（FACS）观察感染情况。通过流式细胞分选仪无菌条件下筛选出表达GFP的EL4细胞．进行大规模扩增培养并对转染前后的细胞进行生物特性分析和比较。在体外用活体成像技术（in vivo imaging）评价其稳定发光能力。C57BL／6小鼠皮下和尾静脉接种发光细胞，构建淋巴瘤小鼠模型，活体内观察肿瘤的生长和转移情况。结果慢病毒载体系统转染293T细胞24h后在荧光显微镜下观察到GFP表达，病毒滴度测定为8．4×10^7 Tu／ml。感染EL4细胞后，荧光显微镜下观察到GFP的表达，FACS分析转导效率为96．5％。筛选转染后的稳定克隆并扩增细胞，分析其生物学特性与EL4细胞无明显差异（P〉0．05）。活体成像系统分析感染后的EL4细胞释放的光子量与细胞数量成正相关（R^2=0.9896）。利用活体成像观察到了皮下移植瘤的生长，并且光子强度随肿瘤的增大而增强；尾静脉接种小鼠后通过活体成像，能在不同时期动态观察到肿瘤的组织、器官转移。结论我们成功制备了荧光素酶标记的EL4细胞并且构建了生物发光淋巴瘤动物模型。该模型可以非侵入性地动态检测活体内白血病／淋巴瘤的演进过程，为示踪肿瘤体内生长、转移，研究肿瘤发展机制及最佳治疗策略的选择提供了新的手段和工具。Objective To construct the murine T-cell leukemia/lymphoma cell lines （EIA） stably expressing firefly luciferase and to evaluate their biolumineseent ability in cell level; to establish bioluminescence lymphoma animal models and to observe the growth and metastasis of tumor in whole body level. Methods Human kideny 293T cells were co-transfected with the four plasmid system using cationic liposome method and the expression of GFP was observed under fluorescence microscope after 24 hours and to monitor transfection efficiency. The viral particles were collected and concentrated after transfection 72 hours. The EIA cells were infected at multiplicity of infection of 8 with the concentrated superuatant containing the virus with the firefly luciferase gene and green fluorescent protein gene. The expression of GFP in EL4 cells was observed by fluorescence microscopy and fluorescence activated cell sorting （FACS）. The EL4 ceils expressing GFP were screened under sterile condition by fluorescence activated cell sorting,then were cultured and amplified, and biologic characteristics of EL4 and EIA-GFP-luc2 ceils were analyzed and compared. The bioluminescent ability of stably luciferase,expressing cells was evaluated by in vivo imaging technology. The murine biol lymphoma models were established by inoculating female C57BL/6 mice with EL4-GFP-luc2 cells through subcutaneous and caudal vein injection, and the growth and metastasis of lymphoma was evaluated in vivo. Results The expression of GFP was observed under fluorescence microscope after transfection 24 hours and the viral titer was 8.4×107 TU/ml. The expression of GFP was also evident in EIA cells and the transduction efficacy was 96.5%. Biologic characteristics of EL4 and EIA-GFP-luc2 ceils showed no significant differences （P〉0.05）. Biol was correlated to tumor cell number and luciferase was stable over time in vitro （R2=0.9896）. Subcutaneous implantation tumor growth of EL4-GFP-luc2 can be observed through in vivo imaging system and photon inten

关 键 词：慢病毒载体 荧光素酶基因 小鼠淋巴瘤细胞EL4 生物发光 

分 类 号：R979.1[医药卫生—药品]