小鼠脾源性内皮祖细胞培养及7．0T磁共振单细胞成像初探

Single Fe2O3-PLL labeled mouse spleen-derived endothelial progenitor cell detection by 7. 0T MR system

目的建立一种小鼠脾源性内皮祖细胞的培养方法，探讨7．0T磁共振（7．0TMR）系统对磁标记单细胞成像的可行性。方法密度梯度离心法分离脾脏单个核细胞，诱导培养得到内皮祖细胞（EPC）。免疫化学、荧光染色鉴定细胞特性。Fe2O3-PLL标记细胞，普鲁士蓝染色，MTT法评价Fe2O3-PLL对细胞生长的影响，邻菲哕啉法定量分析细胞内铁含量。7．0TMR不同序列体外单细胞成像。结果小鼠脾脏单个核细胞经体外诱导培养呈内皮细胞形态，表达EPC表面标志物CD31、CD34、vWF，显示内吞乙酰化低密度脂蛋白（acLDL）、结合凝集素1（UEA-1）等内皮细胞的功能。MTT检测Fe2O3-PLL标记对细胞增殖活力影响不明显，标记组与未标记组吸光度（A）值比较第4天至第7天差异均无统计学意义（第4天，t=2．81；第5天，t=-1．87；第6天，t=-0．298；第7天，t=-0．115；P均〉0．05）。磁标记单细胞能够在7．0TMR检测中成像，自旋回波序列（MSME）图像中标记细胞显示灰度高于梯度回波序列（2D-FLASH），MSME序列图像标记细胞平均导致信号缺失体素个数为20．2个，而2D—FLASH序列图像为30．1个，差异具有统计学意义（t=15．2，P〈0．05）。结论小鼠脾脏单个核细胞可诱导分化为EPC。在7．0TMR检测中Fe2O3-PLL标记的EPC能够实现体外单细胞成像。

Objective To explore the feasibility of Single F203-PLI, labeled mouse spleen-derived endothelial progenitor cells （EPCs） detection by 7.0T MR system. Methods Mononuclear cells （MNCs） were isolated from mouse spleen by density gradient centrifugation and EPCs were obtained by the different adherence of cells. Immunocytochemistry and fluorescent staining were performed to identify EPCs. The EPCs were labeled with Fe2O3-PLL and the intracellular iron was identified with prussian blue staining. MTF assay was assessed to evaluate proliferation of Fe2O3-PLL labeled EPCs, The cells underwent MR imaging with different sequences. Results Cultured in vitro, mouse spleen-derived MNCs resulted in EC-like morphology. These cells expressed EPCs-specific antigens, such as CD31, CD34 and vWF, and had the ability to incorporate ac-LDL and bind UEA-1. Between Fe2O3-PLL labeled EPCs and unlabeled cells, MTT value of light absorption had no statistical significant difference （day4 t = 2.81, day5 t = - 1.87, day6 t = -0.298 ,day7 t = -0. 115 ,all P 〉0.05）. The signal void induced by labeled single cell is 20.2 pixels in MSME sequence, and 20.2 pixels in 3D-FLASH sequence （t = 15.2, P 〈 0.05）. Single cell could be detected by 7.0 T MR system. Conclusion MNCs isolated from mouse spleen can differentiate into endothelial cells in vitro and have the specific property of stem cells. The mouse spleen-derived EPCs can be labeled with Fe2O3-PLL efficiently. The labeled EPCs can be imaged as dispersed single cells.