超顺磁性氧化铁纳米粒子标记内皮祖细胞靶向肝癌的MR实验研究

Magnetic resonance tracking of endothelial progenitor cells labeled with superparamagnetic iron oxide homing to the site of hepatoma

目的采用小动物专用高场MR（7．0T）观察超顺磁性氧化铁纳米粒子（SPIO）标记的骨髓内皮祖细胞（EPCs）归巢至肿瘤局部的情况。方法抽取荷瘤鼠腹腔积液，直接注射法种植于15只裸鼠肝脏，制作小鼠移植性H22HCC模型；取C57BIM6小鼠骨髓分离培养EPCs，用25μg/ml纳米铁复合物（SPIO-PLL）对第三代EPCs进行标记；利用MTT比色实验、流式细胞分析检测磁性标记对细胞生长、凋亡的影响。模型制作第3天将标记细胞经尾静脉移植至模型鼠（n=15）及对照组（n=6）体内，移植后第1、3、7天应用MR观察肿瘤信号改变情况。行铁蓝染色、CD31免疫组织化学检查，并将MR所见与病理切片行对照分析。采用两样本t检验分析数据。结果模型制作3d后。15只裸鼠肝脏均可见肿瘤病灶；MTT比色实验、流式细胞分析示25μg／mlSPIO—PLL标记细胞后，其生长曲线（t=0．281，P〉0．05）、凋亡与未标记细胞间差异未见统计学意义[早期凋亡率：（12．31±1．43）％与（11．57±1．24）％，晚期凋亡率：（0．55±0．07）％与（0．49±0．05）％；t=0．967和1．060，均P〉0．05]；模型组小鼠细胞移植后第3天，MR成像可见信号改变（4／5），低信号呈斑片、条带状，主要位于肿瘤病灶边缘；第7天信号改变程度减低。病理证实标记细胞归巢至肿瘤周围或肿瘤局部。对照组小鼠MR成像未见明显低信号改变。结论EPCs可归巢至肿瘤局部或周围，高场MR可对其进行动态观察。

Objective To track the migration and incorporation of intravenously injected, magnetically labeled endothelial progenitor cells （EPCs） from mouse bone marrow into the blood vessels in a rapid- ly growing HCC model by microMR （7.0 T）. Methods This study was approved by the Institutional Com- mittee on Animal Research. H22 hepatic ascitic cancer cells was directly injected into the left liver lobe of BALB/c nude mice （n= 15）. EPCs derived from bone marrow of C57BL/6 mice were isolated and cultured. The third passage EPCs were collected and labeled with 25 μg/ml superparamagnetic iron oxide （SPIO） and poly-l-lysine （PLL） complex （SPIO-PLL）. MTT assay and flow cytometry were used to evaluate the difference of growth curve and apoptosis between labeled and unlabeled EPCs. EPCs labeled with SPIO-PLL were injected into mice via tail vein in experiment group （on the 3rd day after establishing HCC model） （n= 15） and control group （n = 6）. The signal changes of tumor （the 1st, 3rd and 7th day after transplantation） were observed by microMR. Prussian blue staining and immunohistochemistry staining of CD3I were performed. MRI findings were confirmed by histomorphology. Two-sample t test was used to analyze the data. Results Single tumor was showed in the liver of all mice 3 d after establishing models. Labeling with SPIO- PLL at a concentration of 25 μg/ml did not alter cell growth curve （measured by MTr assay; t=0.281, P〉 0.05） and cell apoptosis （analyzed by flow cytometry）. The apoptosis rates of SPIO-PLL labeled and unlabled EPCs were （12.31±1.43）% and （11.57±1.24）% in early stage, and （0.55±0.07）% and （0.49±0. 05）% in late stage. No significant differences were observed between them （t=0.967, 1.060; both P〉 0. 05）. Migration and incorporation of transplanted and labeled cells into tumor were documented with in vivo microMR as low signal intensity at the tumor periphery as early as the 3rd day after EPCs administration in preformed tumors （4/5）. Prussian blue staining showed iron-positive cells at the sites corresponding to low signal intensity on MRI. The positive cells expressing CD31 existed in intratumoral and peritumoral vessels. There was no signal change in control group at all time points. Conclusions MRI can demonstrate the incorporation of magnetic labeled mouse EPCs into the implanted hepatoma. It may be helpful for early diagnosis and therapy of liver tumor.