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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- 第三军医大学学报 2019年41卷第12期 页码：1117-1124
- 作者机构：
1.400014 重庆，重庆医科大学附属儿童医院:放射科
2.400014 重庆，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
- 作者简介：
[蔡金华，E-mail:664662256@qq.com CAI Jinhua, E-mail: 664662256@qq.com
- 基金信息：国家自然科学基金面上项目(81771892)
DOI：10.16016/j.1000-5404.201812082
中图分类号： R394-33;R445.2;R730.4收稿：2018-12-14，
修回：2019-01-31，
纸质出版：2019-06-30
- 稿件说明：
移动端阅览
孙君, 蔡金华, 秦勇, 等. 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[J]. 第三军医大学学报, 2019,41(12):1117-1124.
Jun SUN, Jinhua CAI, Yong QIN, et al. 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[J]. Journal of Third Military Medical University, 2019, 41(12): 1117-1124.孙君, 蔡金华, 秦勇, 等. 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[J]. 第三军医大学学报, 2019,41(12):1117-1124. DOI： 10.16016/j.1000-5404.201812082.
Jun SUN, Jinhua CAI, Yong QIN, et al. 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[J]. Journal of Third Military Medical University, 2019, 41(12): 1117-1124. DOI： 10.16016/j.1000-5404.201812082.
摘要
目的
2
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
2
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
2
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
2
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。
Abstract
Objective
2
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
2
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
2
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-PEG3-FTH1 cells or TMSCs.
In vitro
MRI showed obviously decreased T
2
signal in TMSCs-PEG3-FTH1 cells as compared with MSCs-PEG3-FTH1 cells.
Conclusion
2
PEG3 promoter can drive the specific expression of FTH1 that allows MRI detection in rat MSCs after malignant transformation.
关键词
Keywords
references
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