PEG3启动子启动铁蛋白报告基因在干细胞瘤变时特异表达及体外磁共振成像  

Specific expression of reporter gene FTH1 driven by progression elevated gene-3 promoter in malignant transformation of rat mesenchymal stem cells and its detection by in vitro magnetic resonance imaging

作　　者：孙君 蔡金华[1] 秦勇[1] 李晓朦 仇嘉文 SUN Jun;CAI Jinhua;QIN Yong;LI Xiaomeng;QIU Jiawen(Department of Radiology,Chongqing Medical University, Chongqing, 400014, China;Key Laboratory of Child Development and Disorders Cofounded by Chongqing and Ministry of Education, Chongqing Key Laboratory of Pediatrics, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Children’ s Hospital, Chongqing Medical University, Chongqing, 400014, China)

机构地区：[1]重庆医科大学附属儿童医院放射科,重庆400014 [2]重庆医科大学附属儿童医院儿童发育疾病研究省部共建教育部重点实验室,儿科学重庆市重点实验室,重庆市儿童发育重大疾病诊治与预防国际科技合作基地,重庆400014

出　　处：《第三军医大学学报》2019年第12期1117-1124,共8页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81771892)~~

摘　　要：目的探讨利用肿瘤特异性启动子PEG3驱动MRI报告基因铁蛋白重链(ferritin heavy chain 1, FTH1)在干细胞瘤变时特异性表达并进行MRI检测的可行性。方法构建PEG3启动子控制FTH1表达的病毒载体PEG3-FTH1-LV,感染大鼠骨髓间充质干细胞(mesenchymal stem cells, MSCs),获得MSCs-PEG3-FTH1细胞,MSCs和MSCs-CMV-FTH1作为对照,通过与大鼠胶质瘤细胞C6间接共培养2周诱导其瘤变为TMSCs、TMSCs-PEG3-FTH1、TMSCs-CMV-FTH1。观察细胞形态,检测细胞迁移能力、细胞增殖速度及细胞表面抗体CD29、CD34、CD45、CD90的表达,裸鼠皮下成瘤实验验证细胞瘤变,Western blot、普鲁士蓝染色、细胞透射电镜和磁共振成像(MRI)检测细胞诱导前后FTH1基因的表达。结果 TMSCs细胞较MSCs细胞变小,多呈纺锤状,增殖速度加快,迁移能力增强,CD34、CD45表达增加,CD90表达减少(P<0.05),裸鼠皮下成瘤阳性;Western blot结果显示:MSCs、MSCs-PEG3-FTH1、TMSCs细胞不表达或少量表达FTH1蛋白,而MSCs-CMV-FTH1、TMSCs-PEG3-FTH1、TMSCs-CMV-FTH1细胞均大量表达FTH1蛋白(P<0.05);普鲁士蓝染色和细胞透射电镜观察到MSCs-CMV-FTH1、TMSCs-PEG3-FTH1、TMSCs-CMV-FTH1细胞内铁颗粒,而在MSCs、MSCs-PEG3-FTH1、TMSCs细胞内无明显铁颗粒,MRI显示TMSCs-PEG3-FTH1细胞信号较MSCs-PEG3-FTH1细胞信号明显降低。结论 PEG3启动子可驱动报告基因FTH1在干细胞瘤变时特异性表达并能被MRI检测。Objective To explore the feasibility of using progression elevated gene-3(PEG3) promoter, a tumor-specific promoter, to drive the specific expression of the reporter gene ferritin heavy chain 1(FTH1) and the detection of FTH1 expression using in vitro magnetic resonance imaging(MRI) in bone marrow mesenchymal stem cells(MSCs) after malignant transformation. Methods A viral vector carrying the reporter gene FTH1 driven by PEG3 promoter was transfected into rat MSCs to obtain MSCs-PEG3-FTH1 cells, and the MSCs transfected with a viral vector carrying FTH1 driven by the CMV promoter(MSCs-CMV-FTH1 cells) and the MSCs without transfection served as the controls. The non-transfected MSCs, MSCs-PEG3-FTH1 cells, and MSCs-CMV-FTH1 cells were indirectly co-cultured with rat glioma C6 cells for 2 weeks to induce their malignant transformation(TMSCs, TMSCs-PEG3-FTH1 and TMSC-PEG3-FTH1 cells, respectively). The morphology, migration and proliferation, and the cell surface antigens CD29, CD34, CD45 and CD90 of the transformed cells were observed and analyzed, and their capacity of subcutaneous tumor formation was evaluated in nude mice. Western blotting, Prussian blue staining, transmission electron microscopy, and in vitro MRI were used to detect the changes in the expression of FTH1 gene in the cells after the transformation. Results Compared with MSCs, most of TMSCs were small in size with a spindle-like morphology and showed strong proliferation and migration abilities with increased expression of CD34 and CD45, decreased expression of CD90(P<0.05), and an ability of subcutaneous tumor formation in nude mice. Western blotting showed that FTH1 protein was not expressed or expressed at low levels in MSCs, MSCs-PEG3-FTH1 cells and TMSCs, while high FTH1 expression was detected in MSCs-CMV-FTH1, TMSCs-PEG3-FTH1 and TMSCs-CMV-FTH1 cells(P<0.05). Prussian blue staining and transmission electron microscopy identified the presence of iron particles in MSCs-CMV-FTH1, TMSCs-PEG3-FTH1 and TMSCs-CMV-FTH1 cells, but not in MSCs, MSCs-P

关 键 词：PEG3启动子 FTH1报告基因 磁共振成像 骨髓间充质干细胞 细胞转化 肿瘤 

分 类 号：R394[医药卫生—医学遗传学] R445.2[医药卫生—基础医学]