中国医学科学院学报

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中国医学科学院学报

中国医学科学院学报 ›› 2012, Vol. 34 ›› Issue (5): 474-479.doi: 10.3881/j.issn.1000-503X.2012.05.007

• 论著 • 上一篇    下一篇

双模态标记F344大鼠骨髓间充质干细胞移植入大鼠心脏后离体及在体影像示踪

曹剑1,王怡宁1,石新琳1,马国涛2,孔令燕1,薛华丹1,雷晶1,何泳蓝1,金征宇1   

  1. 中国医学科学院 北京协和医学院 北京协和医院 1放射科 2心外科,北京 100730
  • 收稿日期:2012-09-13 修回日期:2012-11-07 出版日期:2012-10-31 发布日期:2012-10-31
  • 通讯作者: 王怡宁 电话:010-69155509,电子邮件:yiningpumc@hotmail.com E-mail:yiningpumc@hotmail.com
  • 基金资助:

    国家自然科学基金(81000629)

In Vivo and In Vitro Imaging Tracing of Dual-labeled Bone Mesenchymal Stem Cells Transplanted into Myocardium of F344 Rats

CAO Jian1, WANG Yi-ning1, SHI Xin-lin1,MA Guo-tao2, KONG Ling-yan1,XUE Hua-dan1, LEI Jing1, HE Yong-lan1, JIN Zheng-yu1   

  1. 1Department of Radiology,2Department of Cardiac Surgery, PUMC Hospital, CAMS and PUMC, Beijing 100730, China
  • Received:2012-09-13 Revised:2012-11-07 Online:2012-10-31 Published:2012-10-31
  • Supported by:

    Supported by the National Natural Sciences Foundation of China (81000629)

摘要:

目的 利用超小超顺磁性氧化铁(USPIO)标记带有红色荧光蛋白(RFP)的F344大鼠骨髓间充质干细胞(BMSCs),探索双模态成像示踪标记干细胞示踪的可行性。方法 使用含USPIO(40 μg Fe/ml)的培养基与BMSCs/RFP共孵育培养24 h,标记后行普鲁士蓝染色、透射电镜检查及台盼蓝染色验证标记的有效性和安全性。实验组(n=8)通过心肌局部注射方式将双标干细胞移植至急性心肌梗死F344大鼠的梗死心肌周围,术后不同时间点(1 d、1周、2周和4周)行磁共振成像和荧光成像,对双标干细胞进行示踪并比较信号强度变化;对照组(n=2)不注射细胞,行磁共振和光学成像。4周后将大鼠麻醉处死后取心脏病理行HE染色、普鲁士蓝染色和荧光成像,观察双标干细胞在心肌内的分布。结果 共孵育培养24 h的方式可以有效、安全地构建USPIO-RFP双标BMSCs干细胞,普鲁士蓝染色显示USPIO标记阳性率为99%,透射电镜提示USPIO颗粒主要位于胞质内溶酶体中。台盼蓝染色显示标记组和阴性对照组活细胞率差异无统计学意义(95.7% 比 96.3%,P>0.05)。心肌局部注射双标干细胞的实验组8只大鼠,磁共振成像在术后4周内均能观察到注射区域信号强度降低,并且信号强度随时间的延长变化无统计学意义(P=0.66);在体荧光成像未能检测到荧光信号,而离体荧光成像检测到心脏表面细胞注射区域有较弱的荧光。病理切片染色显示梗死心肌周围细胞核密度增加,心肌中蓝染颗粒及荧光成像发光分布区域与HE染色中细胞核密度增加区域一致。结论 USPIO-RFP双标干细胞注射至大鼠急性梗死心肌后,在体磁共振成像在一定时间内能够实现对干细胞的示踪成像,离体荧光成像和病理荧光成像可以示踪移植干细胞。

关键词: 红色荧光蛋白, 超小超顺磁性氧化铁, 骨髓间充质干细胞, 荧光成像, 磁共振成像

Abstract:

Objective To investigate the feasibility of in vitro and in vivo magnetic resonance imaging (MRI) and fluorescence imaging tracking of transplanted bone mesenchymal stem cells (BMSCs) dual-labeled with ultrasmall superparamagnetic iron oxide (USPIO) and red fluorescence protein (RFP). Methods BMSCs were incubated with culture medium containing USPIO for 24 hours. The Prussian-blue staining, transmission electron microscopy and trypan-blue staining were used to study the efficacy and safety of labeling. F344 rat model of acute myocardial infarction was established by ligating the left anterior descending coronary artery. The dual-labeled BMSCs were injected into the margin of the infraction myocardium. Then MRI and fluorescence imaging were performed to trace the cells both in vitro and in vivo. Postmortal study was carried out to observe the distribution of transplanted cells in myocardium. Results The percentage of dual-labeled BMSCs reached 99% after co-incubating with USPIO for 24 hours. USPIO particles were mainly located in lysosomes. As demonstrated by trypan-blue staining, there was no significant deference in viability between labeled and unlabeled groups (P>0.05). All dual-labeled transplanted BMSCs showed a significant decreasing signal on MRI, and the signal intensity changes had no significant difference over 4 weeks (P=0.66). In vitro cell tracing with fluorescence imaging of isolated heart from F344 rats was successful,while in vivo cell tracing with fluorescence imaging failed. Prussian blue staining showed that USPIO distributed near the infracted myocardium, corresponding with the fluorescence imaging. Conclusion MRI can be used to trace the dual-labeled BMSCs transplanted into F344 rat hearts in vivo, while fluorescence imaging and pathological fluorescence imaging can trace the transplanted cells in vitro.

Key words: red fluorescence protein, ultrasmall superparamagnetic iron oxide, bone mesenchymal stem cells, fluorescence imaging, magnetic resonance imaging

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