三模态报告基因的构建、体外标记和骨髓间充质干细胞显像的可行性  

Novel triple fusion reporter gene for labeling and tracking human mesenchymal stem cells in vitro

作　　者：秦潇潇[1] 胡晓俊[1] 李征然[1] 陈俊伟[1] 吴春[1] 赖丽莎[2] 张丽娜[1] 谢佩怡[1] 孟晓春[1] 朱康顺[1] 

机构地区：[1]中山大学附属第三医院放射科,广州510630 [2]广州市第一人民医院放射科

出　　处：《中华放射学杂志》2014年第9期767-771,共5页Chinese Journal of Radiology

基　　金：国家自然科学基金（81070349）;广州市科技计划（2013J4100118）

摘　　要：目的 探讨三模态报告基因的构建、体外标记和人骨髓间充质干细胞（hMSC）显像的可行性.方法 通过gateway技术构建同时含有增强型绿色荧光蛋白（EGFP）、荧光素酶（luciferase2）和储铁蛋白（ferritin）为报告基因的慢病毒载体pLenti7.3-ferritin-IRES-luciferase2-SV40-EGFP.基因测序检测基因序列的正确性、慢病毒包装、感染hMSC,建立稳定的hMSC转染细胞系.以未转染hMSC为未转染组,转染hMSC为转染组.2组细胞分别应用荧光显微镜检测细胞中EGFP表达,光学成像系统计数细胞中加入催化底物D-luciferin后Is内发光光子数,普鲁士蓝染色检测铁染色率,利用MR的T2-mapping序列检测细胞的T2值.采用Pearson法分析转染组细胞个数与1s内发光光子数的相关性;采用t检验比较转染组和未转染组之间细胞的铁染色率、MR T2-mapping序列T2值,及转染加铁组与未转染加铁组之间T2值的差异.结果 基因测序显示pLenti7.3-ferritin-IRES-luciferase2-SV40-EGFP基因序列正确.慢病毒成功制备并转染hMSC,建立EGFP＋-hMSC稳定细胞系.荧光显微镜可检测到单个转染组细胞EGFP表达,未转染组细胞中未检测到EGFP的表达.1ml不同浓度2.00×106、1.00× 106、5.00× 105、2.50× 105、1.25×105、6.25×104个/ml转染组细胞悬液产生的光子数分别为7.972× 107、3.061×1 07、1.547×107、7.805×106、4.256× 106、1.411×106,二者浓度呈正相关（r=0.993,P＜0.01）.未转染组细胞中未检测到光学信号.普鲁士蓝染色可见转染组和未转染组中hMSC的铁染色率分别（81.6±5.1）％和（21.6±3.8）％,差异有统计学意义（t=20.97,P＜0.01）.标记细胞MR成像,转染组细胞T2值为（628.0±23.1）ms,未转染组T2值为（646.5±17.2）ms,差异无统计学意义（t=1.286,P=0.246）.转染加铁组T2值为（488.3±10.7） ms,明显低于未转染加铁组T2值[（520.8±21.0） ms],差异有统计学意义（t=2.76,P=0.033）.结论 构建同时含有EGFP、lucifeObjective To evaluate the feasibility of labeling and tracing human bone marrow mesenchymal stem cells （hMSC） by a triple fusion reporter gene imaging.Methods A lentivirus vector,the pLenti7.3-ferritin-IRES-luciferase2-SV40-EGFP,which contained optical reporter gene-enhanced green fluorescent protein （EGFP）,bioluminescent reporter gene-luciferase2 and MR reporter gene-ferritin,was constructed by gateway technology.After the accuracy of gene sequence was validated,the lentivirus was packaged and infected hMSC,and a stable transformed hMSC cell line was established by fluorescence-activated cell sorter.The expression of EGFP was inspected with fluoresence microscope; the bioluminescence photon number was counted by optical imaging system in 1 s after D-luciferin was given; the iron content of the cells was detected by prussian blue iron stain; T2 values of the T2-mapping of MRI were measured.After an extended series of 3 to 5 times repeated experiment,results were compared between transfected hMSC and un-transfected hMSC to explore the efficiency of the labeling and tracking hMSC of the reporter genes.The positive rate of expression of the EGFP,the difference in iron stain and MR signal （T2） between the two groups was measured by t test.The bioluminescence photon detected in two gradient groups was count to set a Pearson correlation analysis.Results The gene sequence of pLenti7.3-ferritin-IRES-luciferase2-SV40-EGFP was confirmed by DNA sequencing analysis.The lentivirus vector was successfully constructed and infected hMSC.A stable transformed hMSC cell line,with triple fusion reporter genes,was successfully established for the follow-up test.Single hMSC expressed EGFP could be detected by fluorescence microscope.The optical signal of 2.00× 106,1.00× 106,5.00× 105,2.50× 105,1.25 × 102,6.25 × 104/ml cells could be detected as 7.972× 107,3.061 × 107,1.547 × 107,7.805 × 106,4.256 × 106,1.411 × 106 bioluminescence photon after D-luciferin was given,and there was linear positive correlation betwe

关 键 词：骨髓间充质干细胞 基因显像 对比研究 

分 类 号：R457.7[医药卫生—治疗学]