纳米磁粒子复合物标记对内皮祖细胞生物学特性影响的实验研究

Influence on bionomics of endothelial progenitor cells labeling with magnetic nanoparticles

目的探讨超顺磁性纳米磁粒子复合物Fe2O3-多聚赖氨酸（PLL）标记外周血内皮祖细胞（EPCs）后对细胞生物学特性的影响，为标记细胞的体内外MRI提供实验基础。方法合成Fe2O3-PLL复合物。分离兔外周血单个核细胞，贴壁法筛选出EPCs，25mg／L的Fe2O3-PLL标记EPCs，普鲁士蓝染色、电子显微镜观察细胞内铁，四氮噻唑蓝（MTT）比色试验比较未标记、标记细胞间生长曲线的差异，流式细胞分析检测标记、未标记细胞的细胞周期变化、细胞凋亡、表面标记物表达情况，实时定量聚合酶链反应（PCR）检测标记、未标记细胞的内皮型一氧化氮合酶（eNOS）、KDR、血管性假血友病因子（vWF）基因在mRNA水平上的差异，激光共聚焦显微镜下观察并分析标记、未标记细胞的Ca^2＋浓度、膜流动性变化。实验所得数据，计量资料多组比较采用方差分析，两组间比较采用两样本t检验。结果Fe2O3-PLL对细胞的标记率接近100％，铁颗粒位于细胞质内。25mg／LFe2O3-PLL标记细胞，其生长曲线、细胞周期[G0—G1期为（93.74±3，52）％]、细胞凋亡[早期凋亡率为（12．89±1．81）％]与未标记细胞[细胞周期为（94．57±3．66）％，细胞凋亡率（11．67±1.18）％]相比，差异无统计学意义（t值分别为0．283、0．977、P值均〉0．05）；eNOS、KDR、vWF基因的相对表达量及CD34、CD106、CD146、KDR等细胞表面标记物的表达水平相比差异也无统计学意义（P值均〉0，05）；对Ca^2＋通道影响小，细胞膜流动性无明显变化。结论25mg／L的Fe2O3-PLL对兔外周血EPCs的标记率接近100％，并且对细胞的生物学特性无明显影响，可用于进一步MRI的研究。

Objective To explore the influence of home synthesize magnetic iron oxide （called Fe2O3-PLL） labeling on peripheral blood endothelial progenitor cells （EPCs） bionomics to provide experimental foundation for MR imaging ex and in vivo. Methods Fe2O3 was incubated with PLL for 2 hours to obtain a complex of Fe2O3-PLL. Rabbit peripheral blood mononuclear cells were isolated and EPCs were selected by adherence method. Fe2O3-PLL was used to label EPCs. Prussian blue stain and electron microscope was used for showing intracellular iron. MTT assay was assessed to evaluate the difference of growth curve between unlabeled and labeled with 25 mg/L Fe2O3-PLL. Flow cytometry was performed to analyze cell cycle, cell apoptosis and the expression of surface markers of labeled and unlabeled cells. Expressions of eNOS, KDR and vWF at mRNA levels among unlabeled and labeled EPCs were detected by real-time polymerase chain reaction. Calcium ion channel and membrane fluidity were observed and analyzed by laser confocal microscopy. Statistical analyses were used with ANOVA and t test. Results Almost 100% cells were labeled by Fe2O3-PLL, iron-containing vesicles were intracytoplasma. There was no statistical difference in cells growth curve, cell life cycle [ （93.74 ± 3.52）%, （94. 57 ± 3.66）% ] and cell apoptosis rate（ 12.89 ± 1.81 ）%, （11.67 ± 1.18 ）% ） between labeling with Fe2O3-PLL at a concentration of 25 mg/L and unlabeled cells（ t =0. 283, P 〉0. 05 ; t =0. 977, P 〉0. 05）. There was also no statistical difference in relative amount of eNOS, KDR and vWF at mRNA levels and the expression of surface phenotypic markers （CD34, CD106, CD146 and KDR） between two groups （P 〉 0. 05）. In addition, Labeling had little influence on calcium ion channel and didn＇t significantly alter cell membrane fluidity. Conelusions The rabbit peripheral blood EPCs can be effective labeled with Fe2O3-PLL and without significant influence on cells bionomics at a low concentration of 25 mg/L. Almost every cell can be labeled and the labeled cells can be used further.