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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(40μg 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。
OBJECTIVE: To investigate the feasibility of dual-modal imaging tracer-labeled stem cell tracing using F344 rat bone marrow mesenchymal stem cells (BMSCs) labeled with red fluorescent protein (RFP) using ultra-small superparamagnetic iron oxide (USPIO). Methods The culture medium containing USPIO (40μg Fe / ml) was co-cultured with BMSCs / RFP for 24 h, labeled with Prussian blue staining, transmission electron microscopy and trypan blue staining to verify the safety and efficacy of the marker. In the experimental group (n = 8), double-labeled stem cells were transplanted into the myocardial infarcted myocardium of F344 acute myocardial infarction rats by intramyocardial injection. At different time points (1 d, 1 week, 2 weeks and 4 weeks) Imaging and fluorescence imaging were performed on double-labeled stem cells and the signal intensity was compared. The control group (n = 2) was injected with no magnetic resonance and optical imaging. After 4 weeks, the rats were anesthetized and sacrificed, and their cardiac pathology was observed by HE staining, Prussian blue staining and fluorescence imaging. The distribution of double-labeled stem cells in myocardium was observed. Results Incubation for 24 h could effectively and safely construct USPIO-RFP double-labeled BMSCs stem cells. Prussian blue staining showed that the positive rate of USPIO was 99%. Transmission electron microscopy suggested that USPIO particles mainly located in the cytoplasmic lysosomes. Trypan blue staining showed that there was no significant difference in the number of viable cells between the labeled group and the negative control group (95.7% vs 96.3%, P> 0.05). Eight rats in the experimental group with double-labeled stem cells injected into the myocardium were observed. Magnetic resonance imaging showed that the signal intensity of the injection area was reduced within 4 weeks after operation, and the signal intensity did not change statistically with time (P = 0.66) ; In vivo fluorescence imaging failed to detect fluorescent signals, whereas in vitro fluorescence imaging detected weaker fluorescence in the injection area of the heart surface cells. Pathological staining showed that the density of nuclei around the infarcted myocardium increased, and the blue staining particles in the myocardium and the luminescence distribution area of fluorescence imaging were consistent with the areas of increased nuclear density in HE staining. CONCLUSIONS: After USPIO-RFP double-labeled stem cells are injected into acute infarcted myocardium in rats, magnetic resonance imaging of tracts can generate tracing images of stem cells within a certain period of time. In vitro fluorescence imaging and pathological fluorescence imaging can trace transplanted stem cells.