超顺磁性氧化铁标记间充质干细胞心肌移植的磁共振成像研究

目的探讨应用磁共振（MR）进行干细胞活体心肌内成像的可行性。方法从猪骨髓抽取、分离、培养间充质干细胞（MSC）,用含超顺磁性氧化铁纳米颗粒（铁羧葡胺）的DMEM培养液孵育24h,并用二脒基苯基吲哚（DAPI）和PKH。进行荧光标记。然后将MSC经心外膜直接注入猪急性梗死心肌内,每头猪注射3点。根据每注射点细胞数量以及细胞是否铁标记,12头猪随机分成4组：2×10^6标记细胞组（n=3）、1×10^6标记细胞组（n=3）、5×10^5标记细胞组（n=3）和1×10^6未标记细胞组（n=3）。细胞移植后20—24h行猪心脏1．5TMR成像,1h后处死并根据MR成像图像切取心肌组织行普鲁士蓝染色和免疫荧光检查。结果（1）体外标记：普鲁士蓝染色见标记MSC内大量蓝色颗粒,标记率为100％,电镜证实含铁囊泡位于胞质内。（2）MR成像：注射铁标记细胞的三组（9头猪）进行活体心脏快速小角度翻转梯度回波（T2·WI—Flash 2d）序列成像,发现移植部位均呈明显的低信号,而且注射点信号缺失区的面积大小与回波时间和移植细胞数量有关;而快速自旋回波（T2WI—FSE）序列成像仅隐约可见低信号区甚至难以辨认,但显示梗死病灶和猪心脏结构比T2·WI-Flash 2d序列清晰。未标记细胞组（3头猪）的9个注射点心肌壁均未显示MR低信号区。（3）组织学检查：MR成像低信号区心肌病理学检查见普鲁士蓝染色、DAPI和PKH26三重阳性细胞存在。结论应用磁共振对超顺磁性氧化铁标记的干细胞进行活体心肌内成像是可行的。

In vivo cardiac magnetic resonance imaging of superparamagnetic iron oxides-labeled mesenchymal stem cells in swines

OBJECTIVE: To explore the feasibility of magnetic resonance imaging (MR) on detecting transplanted nanometer small superparamagnetic iron oxides (SPIO) labeled mesenchymal stem cells (MSCs) in swine model with acute myocardial infarction (MI). METHODS: MSCs isolated from swine were incubated with nanometer SPIO for 24 hours and the third-passage MSCs were labeled with DNA dye 4'-6-diamidino-2-phenylindole (DAPI) and aliphatic red fluorescent dye PKH(26)-GL. Presence of small particles of SPIO in MSCs was assessed by Prussian Blue staining and electron microscopy. Three animals in each group received SPIO-labeled MSCs (5 x 10(5); 1 x 10(6); 2 x 10(6)) and MSCs without SPIO (1 x 10(6)) injections into the infarcted myocardium approximately 1 hour following left anterior descending coronary artery. MRI (1.5-T) was performed 20 to 24 hours post infarction in all animals and the animals were subsequently sacrificed for histology 1 hour post MRI. RESULTS: In vitro Prussian Blue staining and electron microscopy examination revealed numerous iron particles in the cytoplasm of MSCs. Low signal intensity spots with the scanning T(2)(*)WI-Flash 2d sequence were detected in all SPIO-MSCs but not in SPIO-negative-MSCs injected myocardial sites in vivo with the clinical 1.5 T scanner. Prussian blue, DAPI and PKH(26) positive cells were detected histologically in sections corresponding to low signal intensity spots area shown on MRI. CONCLUSION: Magnetically labeled MSCs transplanted in myocardial ischemia area of swine can be visualized in vivo with a clinical 1.5-T MRI and could be used for tracking SPIO-MSCs clinically.